Malate dehyrogenases

Abstract

The present invention relates to a recombinant host cell which is capable of producing a dicarboxylic acid and which comprises a mutant malate dehydrogenase resulting in an increased production of the dicarboxylic acid. The invention also relates to a process for producing a dicarboxylic acid, which method comprises fermenting said recombinant host cell in a suitable fermentation medium and producing the dicarboxylic acid.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No. 16/316,988, filed 10 Jan. 2019, which is a National Stage entry of International Application No. PCT/EP2017/067318, filed 11 Jul. 2017, which claims priority to European Patent Application No. 16179315.3, filed 13 Jul. 2016. Each of these applications is incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE (.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-compliant text file (entitled “2919208-497001_Sequence_Listing_ST25.txt” created on 12 Aug. 2020, and 143,341 bytes in size) is submitted concurrently with the instant application, and the entire contents of the Sequence Listing are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a recombinant host cell capable of producing a dicarboxylic acid, and a method for producing a dicarboxylic acid using said recombinant host cell.

BACKGROUND TO THE INVENTION

The 4-carbon dicarboxylic acids malic acid, fumaric acid and succinic acid are potential precursors for numerous chemicals. For example, succinic acid can be converted into 1,4-butanediol (BDO), tetrahydrofuran, and gamma-butyrolactone. Another product derived from succinic acid is a polyester polymer which is made by linking succinic acid and BDO.

Succinic acid for industrial use is predominantly petrochemically produced from butane through catalytic hydrogenation of maleic acid or maleic anhydride. These processes are considered harmful for the environment and costly. The fermentative production of succinic acid is considered an attractive alternative process for the production of succinic acid, wherein renewable feedstock as a carbon source may be used.

Several studies have been carried out on the fermentative production of C4-dicarboxylic acid in (recombinant) yeast.

EP2495304, for example, discloses a recombinant yeast suitable for succinic acid production, genetically modified with genes encoding a pyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a malate dehydrogenase, a fumarase, a fumarate reductase and a succinate transporter.

Despite the improvements that have been made in the fermentative production of dicarboxylic acid in host cells, such as yeast, there nevertheless remains a need for further improved host cells for the fermentative production of dicarboxylic acids.

SUMMARY OF THE INVENTION

The present invention relates to a recombinant host cell which is capable of producing a dicarboxylic acid and which comprises a mutant polypeptide having malate dehydrogenase (MDH) activity. Surprisingly, it was found that the host cell according to the present invention produces an increased amount of a dicarboxylic acid as compared to the amount of dicarboxylic acid produced by a host cell comprising a reference MDH polypeptide, the reference MDH polypeptide being typically a NAD(H)-dependent malate dehydrogenase (EC 1.1.1.37).

According to the present invention, there is thus provided a recombinant host cell which is capable of producing a dicarboxylic acid and which comprises a nucleic acid sequence encoding a mutant polypeptide having malate dehydrogenase activity, wherein the mutant polypeptide comprises an amino acid sequence which, when aligned with the malate dehydrogenase comprising the sequence set out in SEQ ID NO: 39, comprises one mutation (e.g. one substitution) of an amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39. Said mutant polypeptide having malate dehydrogenase activity may further comprise one or more additional mutations (e.g. substitutions). In particular, the mutant polypeptide having malate dehydrogenase activity may further comprise one or more additional mutations (e.g. substitutions) corresponding to any of amino acids 35, 36, 37, 38, 39 and/or 40 in SEQ ID NO: 39.

According to the present invention, there is also provided a recombinant host cell which is capable of producing a dicarboxylic acid and which comprises a nucleic acid sequence encoding a mutant polypeptide having malate dehydrogenase activity, wherein the mutant polypeptide has an increase in the NADP(H)-relative to NAD(H)-dependent activity as compared to that of a reference MDH polypeptide, the reference MDH polypeptide being typically a NAD(H)-dependent malate dehydrogenase (EC 1.1.1.37). In said embodiment, said mutant polypeptide may be a mutant polypeptide comprising an amino acid sequence which, when aligned with the malate dehydrogenase comprising the sequence set out in SEQ ID NO: 39, comprises one mutation (e.g. one substitution) of an amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39.

The invention also provides:

• • a recombinant host cell according to the present invention, wherein the nucleic sequence encoding the mutant polypeptide having malate dehydrogenase activity is expressed in the cytosol and the mutant polypeptide having malate dehydrogenase activity is active in the cytosol.
  • a recombinant host cell according to the present invention, wherein the recombinant host cell has an active reductive tricarboxylic acid (TCA) pathway from phosphoenol or pyruvate to succinate.
  • a method for the production of a dicarboxylic acid, wherein the method comprises fermenting the recombinant host cell of the present invention under conditions suitable for production of the dicarboxylic acid. The dicarboxylic acid may be succinic acid, malic acid and/or fumaric acid. The method may further comprise recovering the dicarboxylic acid from the fermentation medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets out a schematic depiction of integration of fragments 9 to 12. The hatched parts indicated in fragments 9 to 12 denote the unique homologous overlap regions leading to the recombination events as indicated by the dashed crosses between the homologous regions. The 5′ end of fragment 9 and the 3′ end of fragment 12 (indicated by the grey regions in fragments 9 and 12) are homologous to the YPRCtau3 locus on chromosome 16. Homologous recombination results in integration of fragment 10 and 11 into the YPRCtau3 locus.

FIG. 2 sets out a schematic depiction of integration of fragments 1-8 and fragment 113. The hatched parts indicated in fragments 1-8 and 113 denote the unique homologous overlap regions leading to the recombination events as indicated by the dashed crosses between the homologous regions. Fragment 1 and fragment 113 are homologous to the INT59 locus on chromosome XI, homologous recombination results in integration of fragment 2-8 into the INT59 locus.

FIG. 3 sets out a schematic depiction of integration of fragments 13, 114, 115, 15 and 16. The hatched parts indicated in fragments 13, 114, 115, 15 and 16 denote the unique homologous overlap regions leading to the recombination events as indicated by the dashed crosses between the homologous regions. Fragment 13 and fragment 16 are homologous to the INT1 locus on chromosome XV, homologous recombination results in integration of fragment 114, 115 and 15 into the INT1 locus.

FIG. 4 sets out a schematic depiction of integration of fragments 13, 14, 16 and one of the fragments 17-110. The hatched parts indicated in fragments 13, 14, 16 and fragment 17-110 denote the unique homologous overlap regions leading to the recombination events as indicated by the dashed crosses between the homologous regions. Fragment 13 and fragment 16 are homologous to the INT1 locus on chromosome XV, homologous recombination results in integration of fragment 14 and one of the fragments 17-112 into the INT1 locus.

FIG. 5A: Average malic acid titers measured in the supernatant of production medium after cultivation of SUC-1112 transformants, expressing phosphoenolpyruvate carboxykinase (PCKa), pyruvate carboxylase (PYC2), malate dehydrogenase (MDH3), fumarase (FUMR and fumB), dicarboxylic acid transporter (DCT_02) and transformed with reference malate dehydrogenase (SEQ ID NO: 39) or mutant malate dehydrogenase, which contains mutations as compared to the reference sequence in the amino acid positions as indicated in Table 1. The malic acid titer was measured as described in General Materials and Methods and represents an average value obtained from three independent clones.

FIG. 5B: NADH-specific malate dehydrogenase (MDH) activity of MDH mutants expressed in strain SUC-1112 (see Table 1 for specific mutations). Shown is activity, depicted as Δ A340/min/mg total protein. The value is negative as MDH-dependent NADH oxidation results in a decrease in absorbance at 340 nm. A more negative value indicates more activity. The activity was measured as described in Example 5.

FIG. 5C: NADPH-specific malate dehydrogenase (MDH) activity of MDH mutants expressed in strain SUC-1112 (see Table 1 for specific mutations). Shown is activity, depicted as Δ A340/min/mg total protein. The value is negative as MDH-dependent NADPH oxidation results in a decrease in absorbance at 340 nm. A more negative value indicates more activity. The activity was measured as described in Example 5.

FIG. 5D: Ratio of NADPH:NADH dependent activity of MDH mutants expressed in strain SUC-1112 (see Table 1 for specific mutations). The activity was measured and the ratio was determined as described in Example 5. The dashed line indicates a NADPH:NADH ratio of 1.0.

FIG. 6 sets out a schematic depiction of integration of fragments 116, 117, 118 and fragment 119. The hatched parts indicated in fragments 116, 117, 118 and fragment 119 denote the unique homologous overlap regions leading to the recombination events as indicated by the dashed crosses between the homologous regions. Fragment 116 and fragment 119 are homologous to the INT1 locus on chromosome XV, homologous recombination results in integration of fragment 117 and fragment 118 into the INT1 locus.

FIG. 7 sets out a schematic depiction of integration of fragments 1-5, 124 and fragment 120, 121, 122 or 123. The hatched parts indicated in the fragments denote the unique homologous overlap regions leading to the recombination events as indicated by the dashed crosses between the homologous regions. Fragment 1 and fragment 124 are homologous to the INT59 locus on chromosome XI, homologous recombination results in integration of fragment 2-5 and 120, 121, 122 or 123 into the INT59 locus.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 sets out the nucleotide sequence of fragment 2 (FIG. 2), which includes PEP carboxykinase from Actinobacillus succinogenes codon pair optimized for expression in Saccharomyces cerevisiae.

SEQ ID NO: 2 sets out the nucleotide sequence of fragment 3 (FIG. 2), which includes pyruvate carboxylase (PYC2) from S. cerevisiae codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 3 sets out the nucleotide sequence of the PCR template for fragment 4 (FIG. 2), which includes a KanMX selection marker functional in S. cerevisiae.

SEQ ID NO: 4 sets out the nucleotide sequence of fragment 5 (FIG. 2), which includes a putative dicarboxylic acid transporter from Aspergillus niger codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 5 sets out the nucleotide sequence of fragment 6 (FIG. 2), which includes malate dehydrogenase (MDH3) from S. cerevisiae codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 6 sets out the nucleotide sequence of fragment 7 (FIG. 2), which includes fumarase (fumB) from Escherichia coli codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 7 sets out the nucleotide sequence of fragment 8 (FIG. 2), which includes fumarate reductase from Trypanosoma brucei (FRDg) codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 8 sets out the amino acid sequence of fumarate reductase from Trypanosoma brucei (FRDg).

SEQ ID NO: 9 sets out the nucleotide sequence of the primer used to generate fragment 1 (FIG. 2).

SEQ ID NO: 10 sets out the nucleotide sequence of the primer used to generate fragment 1 (FIG. 2).

SEQ ID NO: 11 sets out the nucleotide sequence of the primer used to generate fragment 2 (FIG. 2).

SEQ ID NO: 12 sets out the nucleotide sequence of the primer used to generate fragment 2 (FIG. 2).

SEQ ID NO: 13 sets out the nucleotide sequence of the primer used to generate fragment 3 (FIG. 2).

SEQ ID NO: 14 sets out the nucleotide sequence of the primer used to generate fragment 3 (FIG. 2).

SEQ ID NO: 15 sets out the nucleotide sequence of the primer used to generate fragment 4 (FIG. 2).

SEQ ID NO: 16 sets out the nucleotide sequence of the primer used to generate fragment 4 (FIG. 2).

SEQ ID NO: 17 sets out the nucleotide sequence of the primer used to generate fragment 5 (FIG. 2).

SEQ ID NO: 18 sets out the nucleotide sequence of the primer used to generate fragment 5 (FIG. 2).

SEQ ID NO: 19 sets out the nucleotide sequence of the primer used to generate fragment 6 (FIG. 2) and fragments 120, 121, 122 and 123 (FIG. 7).

SEQ ID NO: 20 sets out the nucleotide sequence of the primer used to generate fragment 6 (FIG. 2) and fragments 120, 121, 122 and 123 (FIG. 7).

SEQ ID NO: 21 sets out the nucleotide sequence of the primer used to generate fragment 7 (FIG. 2).

SEQ ID NO: 22 sets out the nucleotide sequence of the primer used to generate fragment 7 (FIG. 2).

SEQ ID NO: 23 sets out the nucleotide sequence of the primer used to generate fragment 8 (FIG. 2).

SEQ ID NO: 24 sets out the nucleotide sequence of the primer used to generate fragment 8 (FIG. 2).

SEQ ID NO: 25 sets out the nucleotide sequence of the primer used to generate fragment 13 (FIG. 3).

SEQ ID NO: 26 sets out the nucleotide sequence of the primer used to generate fragment 13 (FIG. 3).

SEQ ID NO: 27 sets out the nucleotide sequence of the primer used to generate fragment 14 (FIG. 4).

SEQ ID NO: 28 sets out the nucleotide sequence of the primer used to generate fragment 115 (FIG. 3) and fragment 14 (FIG. 4).

SEQ ID NO: 29 sets out the nucleotide sequence of the primer used to generate fragment 15 (FIG. 3) and fragments 17 to 110 (FIG. 4).

SEQ ID NO: 30 sets out the nucleotide sequence of the primer used to generate fragment 15 (FIG. 3) and fragments 17 to 110 (FIG. 4).

SEQ ID NO: 31 sets out the nucleotide sequence of fragment 15 (FIG. 3) and fragment 120 (FIG. 7), which includes the nucleotide sequence encoding SEQ ID NO: 39 codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 32 sets out the nucleotide sequence of the primer used to generate fragment 16 (FIG. 3).

SEQ ID NO: 33 sets out the nucleotide sequence of the primer used to generate fragment 16 (FIG. 3).

SEQ ID NO: 34 sets out the nucleotide sequence of fragment 9 (FIG. 1), which includes fumarase from Rhizopus oryzae codon pair optimized for expression in Saccharomyces cerevisiae.

SEQ ID NO: 35 sets out the nucleotide sequence of fragment 10 (FIG. 1), which includes the 5′ part of the Cre-recombinase.

SEQ ID NO: 36 sets out the nucleotide sequence of fragment 11 (FIG. 1), which includes the 3′ part of the Cre-recombinase.

SEQ ID NO: 37 sets out the nucleotide sequence of fragment 12 (FIG. 1), which includes a region homologous to the YPRCtau3 locus.

SEQ ID NO: 38 sets out the nucleotide sequence of the PCR template for fragment 115 (FIG. 3), fragment 14 (FIG. 4) and fragment 117 (FIG. 6), which includes the nourseothricin selection marker.

SEQ ID NO: 39 sets out the amino acid sequence of the malate dehydrogenase (MDH3) protein from S. cerevisiae, lacking the 3 C-terminal peroxisomal targeting sequence.

SEQ ID NO: 40 sets out the nucleotide sequence of the primer used to generate fragment 113 (FIG. 2).

SEQ ID NO: 41 sets out the nucleotide sequence of the primer used to generate fragment 113 (FIG. 2).

SEQ ID NO: 42 sets out the nucleotide sequence of fragment 114 (FIG. 3), which includes the expression cassette of ZWF1 from S. cerevisiae codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 43 sets out the nucleotide sequence of the primer used to generate fragment 114 (FIG. 3).

SEQ ID NO: 44 sets out the nucleotide sequence of the primer used to generate fragment 114 (FIG. 3).

SEQ ID NO: 45 sets out the nucleotide sequence of the primer used to generate fragment 115 (FIG. 3).

SEQ ID NO: 46 sets out the amino acid sequence of the pyruvate carboxylase protein from S. cerevisiae.

SEQ ID NO: 47 sets out the amino acid sequence of phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes, with EGY to DAF modification at position 120-122.

SEQ ID NO: 48 sets out the amino acid sequence of fumarase (fumB) from Escherichia coli.

SEQ ID NO: 49 sets out the amino acid sequence of fumarase from Rhizopus oryzae, lacking the first 23 N-terminal amino acids.

SEQ ID NO: 50 sets out the amino acid sequence of a putative dicarboxylic acid transporter from Aspergillus niger.

SEQ ID NO: 51 sets out the amino acid sequence of isocitrate lyase from Kluyveromyces lactis.

SEQ ID NO: 52 sets out the amino acid sequence of Saccharomyces cerevisiae peroxisomal malate synthase (Mls1) amino acid sequence, lacking the 3 C-terminal peroxisomal targeting sequence.

SEQ ID NO: 53 sets out the amino acid sequence of the malate dehydrogenase (MDH3) protein from S. cerevisiae, including the peroxisomal targeting sequence SKL.

SEQ ID NO: 54 sets out the nucleotide sequence of the primer used to generate fragment 116 (FIG. 6).

SEQ ID NO: 55 sets out the nucleotide sequence of the primer used to generate fragment 116 (FIG. 6).

SEQ ID NO: 56 sets out the nucleotide sequence of the primer used to generate fragment 117 (FIG. 6).

SEQ ID NO: 57 sets out the nucleotide sequence of the primer used to generate fragment 117 (FIG. 6).

SEQ ID NO: 58 sets out the nucleotide sequence of the primer used to generate fragment 119 (FIG. 6).

SEQ ID NO: 59 sets out the nucleotide sequence of the primer used to generate fragment 119 (FIG. 6).

SEQ ID NO: 60 sets out the nucleotide sequence of the primer used to generate fragment 118 (FIG. 6).

SEQ ID NO: 61 sets out the nucleotide sequence of the primer used to generate fragment 118 (FIG. 6).

SEQ ID NO: 62 sets out the nucleotide sequence of fragment 118 (FIG. 6) which includes coding sequence for fumarate reductase from Trypanosoma brucei (FRDg) codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 63 sets out the nucleotide sequence of the primer used to generate fragment 124 (FIG. 7).

SEQ ID NO: 64 sets out the nucleotide sequence of fragment 121 (FIG. 7) which includes coding sequence for S. cerevisiae MDH3 mutant MUT_014 codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 65 sets out the nucleotide sequence of fragment 122 (FIG. 7) which includes coding sequence for S. cerevisiae MDH3 mutant MUT_015 codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 66 sets out the nucleotide sequence of fragment 123 (FIG. 7) which includes coding sequence for S. cerevisiae MDH3 mutant MUT_034 codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 67 sets out the amino acid sequence of fumarase from Arabidopsis thaliana.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present specification and the accompanying claims, the words “comprise”, “include” and “having” and variations such as “comprises”, “comprising”, “includes” and “including” are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, “an element” may mean one element or more than one element.

The reductive TCA pathway is one of the primary pathways by which a microorganism can produce dicarboxylic acids. In recent years, it has proven to be the best economic option for the microbial production of dicarboxylic acids, e.g. succinic acid. The reductive TCA pathway includes two reactions which require the consumption of reducing power; i.e. the malate dehydrogenase reaction (reduction of oxaloacetate to malate) and the fumarate reductase reaction (reduction of fumarate to succinate).

Malate dehydrogenase (MDH) catalyzes the reversible conversion of malate to oxaloacetate using NAD or NADP as the cofactor (also collectively referred as NAD(P)). MDH is a rather ubiquitous enzyme and plays crucial roles in many metabolic pathways, including the tricarboxylic acid cycle, amino acid synthesis, gluconeogenesis, maintenance of the oxidation/reduction balance and metabolic stress.

MDHs can be divided into NAD(H)-dependent MDHs (NAD-MDH) (EC 1.1.1.37) and NADP(H)-dependent MDHs (NADP-MDH) (EC 1.1.1.82), according to their preference for cofactors. Most bacterial and archaeal MDHs are NAD-MDHs. Eukaryotic MDH isoforms are all NAD-MDHs, including mitochondrial MDHs, cytosolic MDHs, glyoxysomal MDHs, and peroxisomal MDHs, except for chloroplastic NADP-MDHs, which are required for the transfer of reducing equivalents from chloroplast stroma to cytosol. In the yeast Saccharomyces cerevisiae, three endogenous isoenzymes of malate dehydrogeneases have been identified, namely MDH1, MDH2 and MDH3. They were located in the mitochondria (MDH1), the cytosol (MDH2) and the peroxisome (MDH3) and were all characterized to be NAD(H)-dependent MDHs (EC 1.1.1.37).

The study of NAD(P)-binding domains in the malate dehydrogenase enzyme family revealed a conserved βB-αC motif of the Rossmann fold. The ability of the dehydrogenases to discriminate against NADP(H) lies in the amino acid sequence of this βB-αC motif, which has been predicted to be a principal determinant for cofactor specificity. For example, in the S. cerevisiae peroxisomal NAD-MDH (MDH3), the NAD-binding motif includes amino acid residues 34 to 40 which were found important for cofactor binding and specificity.

In the context of the present invention, it has been surprisingly found that a set of specific mutations in the conserved NAD-binding motif of a polypeptide having MDH activity confers an increased production of a dicarboxylic acid when (over)expressed in a recombinant host cell capable of the production of said dicarboxylic acid. That is to say, (over)expression of said mutant polypeptide having MDH activity in a recombinant host cell typically leads to increased production of a dicarboxylic acid as compared to a recombinant host cell which (over)expresses a reference MDH polypeptide; the “reference MDH polypeptide” being typically a NAD-MDH (EC 1.1.1.37). Concomitantly, it has been shown that said mutant polypeptide having at least one mutation in the conserved NAD-binding motif has an increase in the NADP(H)-relative to NAD(H)-dependent activity as compared to that of said reference MDH polypeptide. Surprisingly, the inventors of the present invention have further shown that the NADP(H)-dependent activity does not have to be higher than the NAD(H)-dependent activity to obtain an increase in dicarboxylic acid production.

It is therefore an object of the present invention to provide a recombinant host cell which is capable of producing a dicarboxylic acid and which comprises a mutant polypeptide having malate dehydrogenase (MDH) activity.

In one embodiment, the mutant polypeptide having malate dehydrogenase activity comprises an amino acid sequence which, when aligned with the malate dehydrogenase comprising the sequence set out in SEQ ID NO: 39, comprises one mutation of an amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39. In other words, said mutant polypeptide comprises one mutation of an amino acid residue occurring at a position corresponding to 34 in SEQ ID NO: 39.

In a preferred embodiment of the invention, the mutation of the amino acid corresponding to amino acid 34 (as defined with reference to SEQ ID NO: 39) will be a substitution.

More preferably, the substitution of the amino acid corresponding to amino acid 34 (as defined with reference to SEQ ID NO: 39) will typically be to a small amino acid. Suitable small amino acids include threonine (T), serine (S), glycine (G), alanine (A) and proline (P). Preferred small amino acids are glycine (G) and serine (S).

In the context of the present invention, a “recombinant host cell” or a “genetically modified host cell” is a host cell into which has been introduced, by means of recombinant DNA techniques, a nucleic acid, a nucleic acid construct or a vector comprising a nucleic acid sequence encoding a mutant polypeptide having malate dehydrogenase activity.

Herein, a “mutant polypeptide having malate dehydrogenase (MDH) activity” may be referred to as a “mutant malate dehydrogenase”, “MDH mutant”, “MDH mutant polypeptide”, “mutant”, “mutant polypeptide” or the like.

Herein, the “malate dehydrogenase activity” is the activity converting oxaloacetic acid to malic acid:
Malic acid+acceptor<=>oxaloacetic acid+reduced acceptor

The term “polypeptide” is used herein for chains containing more than about seven amino acid residues. All polypeptide sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, N Y, 2001).

In the context of the present invention, a “mutant” polypeptide is defined as a polypeptide which was obtained by introduction of one or more mutations. Said mutations may be selected from the group of substitutions, additions and deletions. The term “substitution” herein means the replacement of an amino acid residue in the polypeptide sequence with another one. A “mutant” polypeptide, a “mutated” polypeptide and a “genetically engineered” polypeptide have the same meaning and are used interchangeably.

Herein, a “corresponding position” refers to the vertical column in an amino acid sequence alignment between SEQ ID NO: 39 and sequences homologous to SEQ ID NO: 39 corresponding to a specific position in SEQ ID NO:39 and showing the amino acids that occur at this position in the other aligned homologues.

In the context of the invention, a “corresponding mutation” refers to a mutation of an amino acid residue occurring at a “corresponding position” in SEQ ID NO: 39. For example, a “corresponding substitution” refers to a substitution of an amino acid residue occurring at a “corresponding position” in SEQ ID NO: 39 with another amino acid residue.

In some further embodiments, the mutant polypeptide having malate dehydrogenase activity may further comprise one or more additional mutations corresponding to any of amino acids 35, 36, 37, 38, 39 and/or 40 in SEQ ID NO: 39. Said mutations will typically be selected from the group of substitutions, additions and deletions. More preferably, the one or more additional mutations will be a substitution.

The substitution of the amino acid corresponding to amino acid 35 (as defined with reference to SEQ ID NO: 39) will typically be to a small amino acid. Suitable small amino acids include threonine (T), serine (S), glycine (G), alanine (A) and proline (P). Alternatively, the substitution of the amino acid corresponding to amino acid 35 (as defined with reference to SEQ ID NO: 39) will be to a hydrophobic amino acid, such as isoleucine (I). A preferred substitution of the amino acid corresponding to amino acid 35 (as defined with reference to SEQ ID NO: 39) will be to serine (S) or isoleucine (I).

The substitution of the amino acid corresponding to amino acid 36 (as defined with reference to SEQ ID NO: 39) will typically be to a polar amino acid. Suitable polar amino acids include arginine (R), glutamine (Q), Glutamic acid (E) and serine (S). Alternatively, the substitution of the amino acid corresponding to amino acid 36 (as defined with reference to SEQ ID NO: 39) will be to a small amino acid, such as alanine (A) or proline (P). A preferred substitution of the amino acid corresponding to amino acid 36 (as defined with reference to SEQ ID NO: 39) will be to arginine (R), glutamine (Q), glutamic acid (E), serine (S), alanine (A) or proline (P).

The substitution of the amino acid corresponding to amino acid 37 (as defined with reference to SEQ ID NO: 39) will typically be to a small amino acid. Suitable small amino acids include glycine (G), asparagine (N), and alanine (A). Alternatively, the substitution of the amino acid corresponding to amino acid 37 (as defined with reference to SEQ ID NO: 39) will be to a polar amino acid, such as arginine (R) or glutamine (Q). A preferred substitution of the amino acid corresponding to amino acid 37 (as defined with reference to SEQ ID NO: 39) will be to (G), asparagine (N), alanine (A), alanine arginine (R) or glutamine (Q).

The substitution of the amino acid corresponding to amino acid 38 (as defined with reference to SEQ ID NO: 39) will typically be to a small amino acid. Suitable small amino acids include valine (V), threonine (T), serine (S), glycine (G), alanine (A) and proline (P). A preferred substitution of the amino acid corresponding to amino acid 38 (as defined with reference to SEQ ID NO: 39) will be to alanine (A), valine (V), threonine (T) or serine (S).

The substitution of the amino acid corresponding to amino acid 39 (as defined with reference to SEQ ID NO: 39) will typically be to a small amino acid. A suitable small amino acid includes proline (P). Alternatively, the substitution of the amino acid corresponding to amino acid 39 (as defined with reference to SEQ ID NO: 39) will be to a hydrophobic amino acid, such as lysine (K), phenylalanine (F) or leucine (L). Alternatively, the substitution of the amino acid corresponding to amino acid 39 (as defined with reference to SEQ ID NO: 39) will be to a polar amino acid, such as glutamic acid (E). A preferred substitution of the amino acid corresponding to amino acid 39 (as defined with reference to SEQ ID NO: 39) will be to glutamic acid (E), lysine (K), phenylalanine (F), leucine (L) or proline (P).

The substitution of the amino acid corresponding to amino acid 40 (as defined with reference to SEQ ID NO: 39) will typically be to a small amino acid. Suitable small amino acids include glycine (G). Alternatively, the substitution of the amino acid corresponding to amino acid 40 (as defined with reference to SEQ ID NO: 39) will be to a polar amino acid, such as glutamine (Q). A preferred substitution of the amino acid corresponding to amino acid 40 (as defined with reference to SEQ ID NO: 39) will be to glycine (G) or glutamine (Q).

The various types of amino acids above are classified with reference to, for example, Betts and Russell, In Bioinformatics for Geneticists, Barnes and Gray eds, Wiley 2003.

In more detail, in the context of the invention, a mutant polypeptide having malate dehydrogenase activity will comprise G or S at position 34 as defined with reference to SEQ ID NO: 39;

and, optionally

I or S at position 35 as defined with reference to SEQ ID NO: 39; and/or

R, Q, A, E, P or S at position 36 as defined with reference to SEQ ID NO: 39; and/or

A, N, G, R or Q at position 37 as defined with reference to SEQ ID NO: 39; and/or

A, V, T or S at position 38 as defined with reference to SEQ ID NO: 39; and/or

E, K, P, F or L at position 39 as defined with reference to SEQ ID NO: 39; and/or

G or Q at position 40 as defined with reference to SEQ ID NO: 39.

In one specific embodiment, a mutant polypeptide having malate dehydrogenase activity will comprise a small amino acid at position 34 (as defined with reference to SEQ ID NO: 39) and a small or polar amino acid at position 36 (as defined with reference to SEQ ID NO: 39). In said embodiment, a preferred small amino acid at position 34 may be selected from G or S. In said embodiment, a preferred small or polar amino acid at position 36 may be selected from R, Q, A, E, P or S. Optionally, in said embodiment, the mutant polypeptide will comprise

I or S at position 35 as defined with reference to SEQ ID NO: 39; and/or

A, N, G, R or Q at position 37 as defined with reference to SEQ ID NO: 39; and/or

A, V, T or S at position 38 as defined with reference to SEQ ID NO: 39; and/or

E, K, P, F or L at position 39 as defined with reference to SEQ ID NO: 39; and/or

G or Q at position 40 as defined with reference to SEQ ID NO: 39.

The mutant polypeptide having MDH activity may furthermore comprises additional mutations other than the seven positions defined above, for example, one or more additional substitutions, additions or deletions.

The mutant polypeptide having MDH activity may comprise a combination of different types of modification of this sort. The mutant polypeptide having MDH activity may comprise one, two, three, four, least 5, at least 10, at least 15, at least 20, at least 25, at least 30 or more such modifications (which may all be of the same type or may be different types of modification). Typically, the additional modifications may be substitutions.

In yet further embodiments, the mutant polypeptide having malate dehydrogenase activity is as defined with reference to Table 1 (Example 4) and wherein the amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39 is selected from glycine (G) or serine (S). That is to say, the mutant polypeptide may comprise any combination of substitutions as set out in Table 1 as compared to a suitable reference sequence such as that set out in SEQ ID NO: 39, and wherein the amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39 is selected from glycine (G) or serine (5).

Typically, then the mutant polypeptide may comprise the sequence of SEQ ID NO: 39 with one substitution at position 34, and optionally one or more substitutions at 35, 36, 37, 38, 39 and/or 40. That is to say, the mutant polypeptide will have an amino acid other than aspartate at position 34 and optionally an amino acid other than isoleucine at position 35 and/or an amino acid other than arginine at position 36 and/or an amino acid other than alanine at position 37, and/or an amino acid other than alanine at position 38, and/or an amino acid other than glutamate at position 39, and/or an amino acid other than glycine at position 40.

Also, typically, the mutant polypeptide may comprise the sequence of SEQ ID NO: 39 with one substitution at position 34, one substitution at position 36, and optionally one or more substitutions at 35, 37, 38, 39 and/or 40. That is to say, the mutant polypeptide will have an amino acid other than aspartate at position 34, an amino acid other than arginine at position 36, and optionally an amino acid other than isoleucine at position 35 and/or an amino acid other than alanine at position 37, and/or an amino acid other than alanine at position 38, and/or an amino acid other than glutamate at position 39, and/or an amino acid other than glycine at position 40.

In a separate embodiment of the present invention, the mutant polypeptide having malate dehydrogenase activity has an increase in the NADP(H)-relative to NAD(H)-dependent activity as compared to that of a reference MDH polypeptide. In said embodiment, said mutant polypeptide may be a mutant polypeptide comprising an amino acid sequence which, when aligned with the malate dehydrogenase comprising the sequence set out in SEQ ID NO: 39, comprises one mutation (e.g. one substitution) of an amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39. Further embodiments with regard to the amino acid sequence of said mutant polypeptide are as described herein above.

In the context of the invention, a reference polypeptide having malate dehydrogenase activity, also called a “reference MDH polypeptide”, may be NAD-MDH (EC 1.1.1.37). A reference polypeptide having malate dehydrogenase activity may be a malate dehydrogenase from a microbial source, such as a yeast (e.g. Saccharomyces cerevisiae). A malate dehydrogenase having the amino acid sequence set out in SEQ ID NO: 39 may be a suitable reference polypeptide having MDH activity.

The expression “increase in NADP(H)-relative to NAD(H)-dependent activity” herein typically refers to the property of a mutant polypeptide to show an increase in NADP(H)-relative to NAD(H)-dependent activity in comparison to that of a reference MDH polypeptide, for example in comparison to SEQ ID NO: 39. That is to say a mutant polypeptide may show an increase in the ratio of NADP(H)- to NAD(H)-dependent activity in comparison to that of a reference polypeptide. In Example 5, the ratio is also referred as the “NADPH:NADH specificity ratio”.

In the context of the present invention, the terms “NADP(H)-dependent activity” and “NADP(H)-specific activity” have the same meaning herein and are used interchangeably. Same applies for the terms “NAD(H)-dependent activity” and “NAD(H)-specific activity”.

The term “NADP(H)-dependent activity” herein refers to the property of an enzyme to use NADP(H) as the redox cofactor. The NADP(H)-dependent activity of the enzyme may be determined by an enzyme activity assay such as described in Example 5.

The term “NAD(H)-dependent activity” herein refers to the property of an enzyme to use NAD(H) as the redox cofactor. The NAD(H)-dependent activity of the enzyme may be determined by an enzyme activity assay such described in Example 5.

An increased value of the average NADPH:NADH specificity ratio may indicate, for example, a reduced NAD(H)-dependent activity, an increased NADP(H)-dependent activity or a combination of the two. In some cases, an increased value of said ratio may be obtained with a MDH mutant having a similar or increased NAD(H)-dependent activity in comparison to a reference MDH. In the latter cases, it may be that the MDH mutant displays both increased NAD(H)- and NADP(H)-dependent activities.

The mutant MDH polypeptide will typically have modified MDH activity in terms of modified cofactor dependence. This NAD(H)- or NADP(H)-dependent activity may be modified independent from each other, for example decreased, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%. Alternatively, the property may be increased by at least 10%, at least 25%, at least 50%, at least 70%, at least 100%, at least, 200%, at least 500%, at least 700%, at least 1000%, at least 3000%, at least 5000%, or at least 6000%.

In one specific embodiment, the NAD(H)- and NADP(H)-dependent activities of the mutant MDH polypeptide are both increased. The NAD(H)-dependent activity of said mutant MDH polypeptide is increased by at least 10%, at least 25%, at least 50%, at least 70%, at least 100%, at least, 200%, or at least 300%. The NADP(H)-dependent activity of said mutant MDH polypeptide is increased by at least 10%, at least 25%, at least 50%, at least 70%, at least 100%, at least, 200%, at least 500%, at least 700%, at least 1000%, at least 3000%, at least 5000%, or at least 6000%.

In another embodiment, the NAD(H)-dependent activity of the mutant MDH polypeptide is about the same or decreased by at most 5%, at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80% and the NADP(H)-dependent activity of said mutant MDH polypeptide is increased by at least 10%, at least 25%, at least 50%, at least 70%, at least 100%, at least, 200%, at least 500%, at least 700%, at least 1000%, at least 3000%, at least 5000%, or at least 6000%.

In another embodiment, the NADPH:NADH specificity ratio of the mutant MDH polypeptide is increased by at least 10%, at least 50%, at least 100%, at least, 200%, at least 500%, at least 1000%, at least 3000%, at least 5000%, at least 7000%, or at least 8000%.

The percentage decrease or increase in this context represents the percentage decrease or increase in comparison to the reference MDH polypeptide, for example that of SEQ ID NO: 39. It is well known to the skilled person how such percentage changes may be measured—it is a comparison of the activity, for example NAD(H)- or NADP(H)-dependent activity, of the reference MDH and the mutant MDH measured as set out in the Examples.

In the context of the present invention, the MDH mutant polypeptide as described herein above may be a mutant NAD(P)-malate dehydrogenase, such as a mutant mitochondrial NAD-MDH, a mutant cytosolic NAD-MDH, a mutant glyoxysomal NAD-MDH, a mutant peroxisomal NAD-MDH, or a mutant chloroplastic NADP-MDH. That is to say, the mutant polypeptide having malate dehydrogenase activity may be obtained by introduction of one or more mutations in a NAD(P)-malate dehydrogenase, such as a mitochondrial NAD-MDH, cytosolic NAD-MDH, glyoxysomal NAD-MDH, peroxisomal NAD-MDH, or a chloroplastic NADP-MDH (the latter being referred as template MDH polypeptides for introducing said one or more mutations). Preferably, the mutant polypeptide having malate dehydrogenase activity is a mutant NAD(H)-malate dehydrogenase (EC 1.1.1.37). More preferably, the mutant polypeptide having malate dehydrogenase activity is a mutant peroxisomal NAD(H)-malate dehydrogenase. Even more preferably, the mutant polypeptide having malate dehydrogenase activity is a mutant NAD(H)-malate dehydrogenase from a yeast or a fungus. Even more preferably, the mutant polypeptide having malate dehydrogenase activity is a mutant NAD(H)-malate dehydrogenase from a yeast or fungus, such as S. cerevisiae, Torulaspora delbrueckii, Zygosaccharomyces bailiff, Naumovozyma casteffii, Naumovozyma dairenensis, Lachancea lanzarotensis, Zygosaccharomyces rouxii, Kazachstania africana, Candida tropicalis, Kluyveromyces marxianus, Scheffersomyces stipites, Talaromyces mameffei, Rasamsonia emersonii, Aspergillus niger, or Trametes versicolor. The following Uniprot database codes refer to suitable yeast and fungal template MDH polypeptides (http://www.uniprot.org): E7NGH7, G8ZXS3, G0V668, W0VUI8, G0WB63, A0A0W0D4X6, A0A0C7MME9, C5DQ42, C5D145, H2AWW6, A0A090C493, J7R0C8, Q6CJP3, Q759M4, I2H037, C5M546, A7TL95, A0A0L0P3G3, Q6BM17, S8AW17, V5FMV2, A3LW84, A0A109UZS1, G8BVW8, G8BJ12, M3HPK4, A0A093UW53, B8MTP0, A0A0F4YPR0, C8V0H6, W6QNU3, A5DZ33, U1GAT6, G3B7S5, C4JP17, A0A0F8UZY9, Q4WDM0, A0A093UPX3, B8ND04, A0A0M9VRI4, G7XZ98, Q5A5S6, M7S9E4, E4UYX5, A5DE02, A0A0J7B1J5, A0A017SKI1, G8Y7A1, A0A0G2EFQ2, R7S165, I1RFM4, R1EVC8, U4L6K9, A0A0L0P507, W7HM94, A5DGY9, F2QY33, A0A0G2JA24, UPI000462180C, C7Z9W6, E5AAQ2, B2VVR8, A0A0H2RCV1, A8Q524, A0A0E9N879, N1JA02, A0A0D1ZEE3, J5T1X5, W6MY07, C4Y826, G3AJA2, G9N6G5, AOAOK8L6L9, A3GH28, A8P7W6, K5W0T4, G1XT67, A0A0B7K175, B8MTP5, A0A0D1ZAS7, A0A0C3BQC4, K5Y2Q9, A0A0C3DSW4, A0A068S518, W2RPL2, A0A0H5C453, A0A074WKG5, G8JRX4, A0A0U1M134, H6C0V9, A0A0H5BZ30, M2UXR7, A0A0C9YJV6, A0A0C3S6T1, W1Q K02, A0A0C2YFC4, A0A061AJ54, A0A086T183, W2RVT1, UPI0004623914, A0A0C9X7U1, UPI0001F26169, G7E054, A0A0C2S9T3, A0A067NIX1, L8FPM0, G3ALW4, A0A0D0AYX2, G0VJG3, A0A0D2NGY0, C1GLB8, W9X415, A0A0D0CDE8, S7Q8G0, A0A0C9TB51, R7YP89, A0A0C2W4H8, UPI000455FA04, A0A067NL73, A0A067STP4, W9VWVP5, A0A0D7A9T7, A0A0D6R2E1, M2YLX9, G0W7D4, N1QJ61, G4TRY5, FOXJ10, A0A063BQQ6, A0A061HBX5, A0A0A1PCT2, M1WIC4, A8QAQ2, A0A0C3N631, F2QTL7, A0A060S7U3, A0A0L0HTQ9, Q0CKY1, A0A0C2ZJ90, K5W527, I4Y5C3, R7YXZ2, F9XI12, A0A061J968, F9XL74, A0A0D0BEW9, A0A0C9WB72, F7WA21, A8NJ67, M2P8M6, W4KHW3, A0A0L1I2T4, UPI0004449A9D, P83778, C4Y9Q7, A0A0D7BHV7, A0A068RWX9, M2YWQ3, A0A137QV51, A0A0D0B6C2, I1BQQ7, S3DA07, Q4DXL5, G8Y022, A0A0C9W9B3, A0A0L6WJ49, A0A0L9SL52, D5GA85, A0A0N1J4Z6, J7S1G2, A0A0F4X5C6, Q6CIK3, A0A067QNN0, Q0UGT7, F8ND69, U5HIM0, J3NKC7, A0A061ATZ7, A5DSY0, Q9Y750, UPI0004F4119A, A0A086TL69, A0A0J0XSS4, UPI0003F496E1, UPI000455F0EA, S7RXX1, A0A067NAG9, A0A0B7N3M5, E6ZKH0, C8V1V3, A7UFI6, T5AEM1, A0A072PGA9, A0A094EKH6, S8ADX4, G8C073, Q6BXI8, G2R9I6 and U9TUL6. Even more preferably, the mutant polypeptide having malate dehydrogenase activity is a mutant S. cerevisiae peroxisomal NAD-MDH (MDH3).

Additionally, in the recombinant host cell of the invention, the mutant polypeptide having malate dehydrogenase activity may be a mutant of a homologous or heterologous NAD(P)-malate dehydrogenase. In a preferred embodiment, the MDH mutant is a mutant of a homologous NAD(P)-malate dehydrogenase. More preferably, the MDH mutant is a mutant of a homologous NAD(H)-malate dehydrogenase (EC 1.1.1.37). More preferably, the mutant polypeptide having malate dehydrogenase activity is a mutant of a homologous peroxisomal NAD(H)-malate dehydrogenase.

In this context, the term “homologous” or “endogenous” when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organism of the same species, preferably of the same variety or strain.

The term “heterologous” as used herein refers to nucleic acid or amino acid sequences not naturally occurring in a host cell. In other words, the nucleic acid or amino acid sequence is not identical to that naturally found in the host cell.

Preferably, in a recombinant host cell of the present invention, the nucleic sequence encoding said mutant polypeptide having malate dehydrogenase activity is expressed in the cytosol and the mutant polypeptide having malate dehydrogenase activity is active in the cytosol. In some instances, cytosolic expression may be obtained by deletion of a peroxisomal or mitochondrial targeting signal. The presence of a peroxisomal or mitochondrial targeting signal may for instance be determined by the method disclosed by Schluter et al., Nucleid Acid Research 2007, 35, D815-D822. When the MDH mutant is a mutant S. cerevisiae peroxisomal NAD-MDH (e.g. a mutant MDH3), its C-terminal SKL is preferably deleted such that it is active in the cytosol.

Typically, the mutant polypeptide having malate dehydrogenase activity may have at least about 40%, 50%, 60%, 70%, 80% sequence identity with a reference MDH polypeptide, such as the MDH of SEQ ID NO: 53 or SEQ ID NO: 39, for example at least 85% sequence identity with a reference MDH polypeptide, such as at least about 90% sequence identity with a reference MDH polypeptide, at least 95% sequence identity with a reference MDH polypeptide, at least 98% sequence identity with a reference MDH polypeptide or at least 99% sequence identity with a reference MDH polypeptide.

It has been surprisingly found that said mutant MDH polypeptide as described herein above confers an increase in the production of a dicarboxylic acid in a recombinant host cell when said mutant is (over)expressed in said recombinant host cell as compared to the production level of an equivalent recombinant host cell which (over)expresses a reference polypeptide having MDH activity; the “reference MDH polypeptide” being typically a NAD-MDH (EC 1.1.1.37), for example a malate dehydrogenase having an amino acid sequence set out in SEQ ID NO: 39.

Accordingly, there is thus provided a recombinant host cell which is capable of producing or produces a dicarboxylic acid and which comprises a nucleic acid sequence encoding a mutant polypeptide having malate dehydrogenase activity as described herein above.

A recombinant host cell of the invention is capable of producing or produces a dicarboxylic acid, such as malic acid, fumaric acid and/or succinic acid.

The terms “dicarboxylic acid” and “dicarboxylate”, such as “succinic acid” and “succinate”, have the same meaning herein and are used interchangeably, the first being the hydrogenated form of the latter.

Typically, the recombinant host cell of the invention will produce an increased amount of a dicarboxylic acid in comparison to a recombinant host cell expressing a reference MDH polypeptide, for example that of SEQ ID NO: 39. The production of a dicarboxylic acid may be increased, by at least 5%, 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% or more. Production level may be expressed in terms of g/L, so an increase in the production level of a dicarboxylic acid will be evident by higher level of production in terms of g/L.

The recombinant host cell of the invention or a parent of said host cell may be any type of host cell. Accordingly, both prokaryotic and eukaryotic cells are included. Host cells may also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.

A suitable host cell of the invention may be a prokaryotic cell. Preferably, the prokaryotic cell is a bacterial cell. The term “bacterial cell” includes both Gram-negative and Gram-positive microorganisms.

Suitable bacteria may be selected from e.g. Escherichia, Actinobacillus, Anabaena, Caulobactert, Gluconobacter, Mannheimia, Basfia, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Actinomycetes such as Streptomyces and Actinoplanes species. Preferably, the bacterial cell is selected from the group consisting of Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, Actinobacillus succinogenes, Gluconobacter oxydans, Caulobacter crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Pseudomonas putida, Pseudomonas fluorescens, Paracoccus denitrificans, Escherichia coli, Corynebacterium glutamicum, Mannheimia succinoproducens, Basfia succinoproducens, Staphylococcus carnosus, Streptomyces lividans, Streptomyces clavuligerus, Sinorhizobium melioti and Rhizobium radiobacter.

A host cell according to the invention may be a eukaryotic host cell. Preferably, the eukaryotic cell is a mammalian, insect, plant, fungal, or algal cell. More preferably, the eukaryotic cell is a fungal cell. A suitable fungal cell may for instance belong to genera Saccharomyces, Schizosaccharomyces, Aspergillus, Penicillium, Pichia, Kluyveromyces, Yarrowia, Candida, Hansenula, Humicola, Pichia, Issatchenkia, Kloeckera, Schwanniomyces, Torulaspora, Trichosporon, Brettanomyces, Rhizopus, Zygosaccharomyces, Pachysolen or Yamadazyma. A fungal cell may for instance belong to a species of Saccharomyces cerevisiae, S. uvarum, S. bayanus S. pastorianus, S. carlsbergensis, Aspergillus niger, Penicillium chrysogenum, Pichia stipidis, P. pastoris, Kluyveromyces marxianus, K. lactis, K. thermotolerans, Yarrowia lipolytica, Candida sonorensis, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis, C. kruisei, C. glabrata, Hansenula polymorpha, Issatchenkia orientalis, Torulaspora delbrueckii, Brettanomyces bruxellensis, Rhizopus oryzae or Zygosaccharomyces bailii. In one embodiment, a fungal cell of the present invention is a yeast, for instance belonging to a Saccharomyces sp., such as a Saccharomyces cerevisiae.

Examples of specific host yeast cells include C. sonorensis, K. marxianus, K. thermotolerans, C. methanesorbosa, Saccharomyces bulderi (S. bulden), P. kudriavzevii, I. orientalis, C. lambica, C. sorboxylosa, C. zemplinina, C. geochares, P. membranifaciens, Z. kombuchaensis, C. sorbosivorans, C. vanderwaltii, C. sorbophila, Z. bisporus, Z. lentus, Saccharomyces bayanus (S. bayanus), D. castellii, C, boidinii, C. etchellsii, K. lactis, P. jadinii, P. anomala, Saccharomyces cerevisiae (S. cerevisiae), Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation), Candida ethanolica, P. deserticola, P. membranifaciens, P. fermentans and Saccharomycopsis crataegensis (S. crataegensis). Suitable strains of K. marxianus and C. sonorensis include those described in WO 00/71738 A1, WO 02/42471 A2, WO 03/049525 A2, WO 03/102152 A2 and WO 03/102201A2. Suitable strains of I. orientalis are ATCC strain 32196 and ATCC strain PTA-6648. In the invention, the host cell may be a Crabtree negative as a wild-type strain. The Crabtree effect is defined as the occurrence of fermentative metabolism under aerobic conditions due to the inhibition of oxygen consumption by a microorganism when cultured at high specific growth rates (long-term effect) or in the presence of high concentrations of glucose (short-term effect). Crabtree negative phenotypes do not exhibit this effect, and are thus able to consume oxygen even in the presence of high concentrations of glucose or at high growth rates.

The eukaryotic cell may be a filamentous fungal cell. Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocaffimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma. Preferred filamentous fungal strains that may serve as host cells belong to the species Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtora thermophyla. Reference host cells for the comparison of fermentation characteristics of transformed and untransformed cells, include e.g. Aspergillus niger CBS120.49, CBS 513.88, Aspergillus oryzae ATCC16868, ATCC 20423, IFO 4177, ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, Aspergillus fumigatus AF293 (CBS101355), P. chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Thielavia terrestris NRRL8126, Talaromyces emersonii CBS 124.902, Rasamsonia emersonii CBS393.64, Acremonium chrysogenum ATCC 36225, ATCC 48272, Trichoderma reesei ATCC 26921, ATCC 56765, ATCC 26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense ATCC44006 and derivatives of all of these strains.

A more preferred host cell belongs to the genus Aspergillus, more preferably the host cell belongs to the species Aspergillus niger. When the host cell according to the invention is an Aspergillus niger host cell, the host cell preferably is CBS 513.88, CBS124.903 or a derivative thereof.

In a preferred embodiment, a host cell according to the invention is a yeast cell selected from the group consisting of Candida, Hansenula, Issatchenkia, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strains, or a filamentous fungal cell selected from the group consisting of filamentous fungal cells belong to a species of Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces, Rasamsonia, Thielavia, Fusarium or Trichoderma.

A host cell of the invention may be any wild type strain producing a dicarboxylic acid. Furthermore, a suitable host cell may be a cell which has been obtained and/or improved by subjecting a parental or wild type cell of interest to a classical mutagenic treatment or to recombinant nucleic acid transformation. Thus, a suitable host cell may already be capable of producing the dicarboxylic acid. However, the cell may also be provided with a homologous or heterologous expression construct that encodes one or more polypeptides involved in the production of the dicarboxylic acid.

Accordingly, in some embodiments, a recombinant host cell of the invention may comprise a MDH mutant polypeptide and an active reductive tricarboxylic acid (TCA) pathway from phosphoenolpyruvate or pyruvate to succinate.

Accordingly, in addition to a nucleic acid encoding a MDH mutant polypeptide, a host cell of the invention may comprise a nucleotide sequence comprising sequence encoding one or more of a pyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a phosphoenolpyruvate carboxylase, a malate dehydrogenase, a fumarase, an isocitrate lyase, a malate synthase, a fumarate reductase and/or a dicarboxylic acid transporter. Preferably, one or more such enzymes are (over)expressed and active in the cytosol.

Thus, a recombinant host cell of the invention may overexpress a suitable homologous or heterologous nucleotide sequence that encodes a endogenous and/or heterologous enzyme that catalyzes a reaction in the cell resulting in an increased flux towards a dicarboxylic acid such malic acid, fumaric acid and/or succinic acid.

A recombinant host cell of the invention may overexpress an endogenous or heterologous nucleic acid sequence as described herein below.

A recombinant host cell of the invention may comprise a genetic modification with a pyruvate carboxylase (PYC), that catalyses the reaction from pyruvate to oxaloacetate (EC 6.4.1.1). The pyruvate carboxylase may for instance be active in the cytosol upon expression of the gene. For instance, the host cell overexpresses a pyruvate carboxylase, for instance an endogenous or homologous pyruvate carboxylase is overexpressed. The recombinant fungal host cell according to the present invention may be genetically modified with a pyruvate carboxylase which has at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 46.

Preferably, the recombinant host cell expresses a nucleotide sequence encoding a phosphoenolpyruvate (PEP) carboxykinase in the cytosol. Preferably a nucleotide sequence encoding a PEP carboxykinase is overexpressed. The PEP carboxykinase (EC 4.1.1.49) preferably is a heterologous enzyme, preferably derived from bacteria, more preferably the enzyme having PEP carboxykinase activity is derived from Escherichia coli, Mannheimia sp., Actinobacillus sp., or Anaerobiospirillum sp., more preferably Mannheimia succiniciproducens. A gene encoding a PEP carboxykinase may be overexpressed and active in the cytosol of a fungal cell. Preferably, a recombinant fungal cell according to the present invention is genetically modified with a PEP carboxykinase which has at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 47.

Preferably, the recombinant host cell expresses a nucleotide sequence encoding a phosphoenolpyruvate (PEP) carboxylase in the cytosol. Preferably a nucleotide sequence encoding a PEP carboxylase is overexpressed. The PEP carboxylase (EC 4.1.1.31) preferably is a heterologous enzyme, preferably derived from bacteria.

In one embodiment, the recombinant host cell is further genetically modified with a gene encoding a malate dehydrogenase (MDH) active in the cytosol upon expression of the gene. Cytosolic expression may be obtained by deletion of a peroxisomal targeting signal. The malate dehydrogenase may be overexpressed. A cytosolic MDH may be any suitable homologous or heterologous malate dehydrogenase, catalyzing the reaction from oxaloacetate to malate (EC 1.1.1.37), for instance derived from S. cerevisiae.

Preferably, the MDH is S. cerevisiae MDH3, more preferably one which has a C-terminal SKL deletion such that it is active in the cytosol. Preferably, the recombinant fungal cell according to the present invention comprises a nucleotide sequence encoding a malate dehydrogenase that has at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 39.

In another embodiment, the recombinant host cell of the present disclosure is further genetically modified with a gene encoding a fumarase, that catalyses the reaction from malic acid to fumaric acid (EC 4.2.1.2). A gene encoding fumarase may be derived from any suitable origin, preferably from microbial origin, for instance a yeast such as Saccharomyces or a filamentous fungus, such Rhizopus oryzae, or a bacterium such a Escherichia coli. The host cell of the present disclosure may overexpress a nucleotide sequence encoding a fumarase. The fumarase may be active in the cytosol upon expression of the nucleotide sequence, for instance by deleting a peroxisomal targeting signal. It was found that cytosolic activity of a fumarase resulted in a high productivity of a dicarboxylic acid by a fungal cell.

Preferably, the recombinant host cell of the present invention overexpresses a nucleotide sequence encoding a fumarase that has at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 67.

In another embodiment, the recombinant host cell is genetically modified with any suitable heterologous or homologous gene encoding a NAD(H)-dependent fumarate reductase, catalyzing the reaction from fumarate to succinate (EC 1.3.1.6). The NAD(H)-dependent fumarate reductase may be a heterologous enzyme, which may be derived from any suitable origin, for instance bacteria, fungi, protozoa or plants. A fungal cell of the present disclosure comprises a heterologous NAD(H)-dependent fumarate reductase, preferably derived from a Trypanosoma sp, for instance a Trypanosoma brucei. In one embodiment, the NAD(H)-dependent fumarate reductase is expressed and active in the cytosol, for instance by deleting a peroxisomal targeting signal. The host cell may overexpress a gene encoding a NAD(H)-dependent fumarate reductase.

Preferably, the recombinant host cell according to the present invention is genetically modified with a NAD(H)-dependent fumarate reductase, which has at least at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8. Also preferably, the host cell according to the present invention is genetically modified with a variant polypeptide having fumarate reductase activity as disclosed in WO2015/086839.

In another embodiment, the recombinant host cell of the invention expresses a nucleotide sequence encoding a dicarboxylic acid transporter protein. Preferably the dicarboxylic acid transporter protein is overexpressed. A dicarboxylic acid transporter protein may be any suitable homologous or heterologous protein. Preferably the dicarboxylic acid transporter protein is a heterologous protein. A dicarboxylic acid transporter protein may be derived from any suitable organism, preferably from yeast or fungi such as Schizosaccharomyces pombe or Aspergillus niger. Preferably, a dicarboxylic acid transporter protein is a dicarboxylic acid transporter/malic acid transporter protein, eg. from Aspergillus niger which at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 50.

The recombinant host cell may further comprise a genetic modification with a gene encoding an isocitrate lyase (EC 4.1.3.1), which may be any suitable heterologous or homologous enzyme. The isocitrate lyase may for instance be obtained from Kluyveromyces lactis or Escherichia coli.

The recombinant host according to the present invention is genetically modified with a isocitrate lyase which has at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 51.

The recombinant host cell may further comprise a genetic modification with a malate synthase (EC 2.3.3.9). The malate synthase may be overexpressed and/or active in the cytosol, for instance by deletion of a peroxisomal targeting signal. In the event the malate synthase is a S. cerevisiae malate synthase, for instance the native malate synthase is altered by the deletion of the SKL carboxy-terminal sequence.

The recombinant host cell of the present invention is genetically modified with a malate synthase which at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 52.

In another embodiment, the recombinant host cell of the invention disclosed herein comprises a disruption of a gene a pyruvate decarboxylase (EC 4.1.1.1), catalyzing the reaction from pyruvate to acetaldehyde.

In another embodiment, the recombinant host cell of the invention may comprise a disruption of a gene encoding an enzyme of the ethanol fermentation pathway. A gene encoding an enzyme of an ethanol fermentation pathway, may be pyruvate decarboxylase (EC 4.1.1.1), catalyzing the reaction from pyruvate to acetaldehyde, or alcohol dehydrogenase (EC 1.1.1.1), catalyzing the reaction from acetaldehyde to ethanol. Preferably, a host cell of the invention comprises a disruption of one, two or more genes encoding an alcohol dehydrogenase. In the event the fungal cell is a yeast, e.g. S. cerevisiae, the yeast preferably comprises a disruption of one or more alcohol dehydrogenase genes (adh1 adh2, adh3, adh4, adh5, adh6).

Alternatively or in addition, the recombinant host cell of the invention may comprise at least one gene encoding glycerol-3-phosphate dehydrogenase which is not functional. A glycerol-3-phosphate dehydrogenase gene that is not functional is used herein to describe a eukaryotic cell, which comprises a reduced glycerol-3-phosphate dehydrogenase activity, for instance by mutation, disruption, or deletion of the gene encoding glycerol-3-phosphate dehydrogenase, resulting in a decreased formation of glycerol as compared to a wild-type cell. In the event the fungal cell is a yeast, e.g. S. cerevisiae, the yeast preferably comprises a disruption of one or more glycerol-3-phosphate dehydrogenase genes (gpd1, gpd2, gut2).

Alternatively or in addition to the above, the recombinant host cell of the invention may comprise at least one gene encoding a mitochondrial external NADH dehydrogenase which is not functional. A mitochondrial external NADH dehydrogenase gene that is not functional is used herein to describe a eukaryotic cell, which comprises a reduced NADH dehydrogenase activity, for instance by mutation, disruption, or deletion of the gene encoding the mitochondrial external NADH dehydrogenase. In the event the fungal cell is a yeast, e.g. S. cerevisiae, the yeast preferably comprises a disruption of one or more mitochondrial external NADH dehydrogenase genes (nde1, nde2).

Alternatively or in addition to the above, the recombinant host cell of the invention may comprise at least one gene encoding an aldehyde dehydrogenase which is not functional. An aldehyde dehydrogenase gene that is not functional is used herein to describe a eukaryotic cell, which comprises a reduced aldehyde dehydrogenase activity, for instance by mutation, disruption, or deletion of the gene encoding the aldehyde dehydrogenase. In the event the fungal cell is a yeast, e.g. S. cerevisiae, the yeast preferably comprises a disruption of one or more aldehyde dehydrogenase genes (ald2, ald3, ald4, ald5, ald6).

Preferably, the recombinant host cell of the present invention is a recombinant fungal cell. More preferably, the host cell of the present invention is a recombinant yeast cell. Preferred embodiments of the recombinant fungal cell or recombinant yeast cell are as described herein above for the recombinant host cell.

In some embodiments of the invention, the recombinant host cell is a recombinant yeast cell which is capable of producing a dicarboxylic acid as described herein above and which comprises a nucleic acid sequence encoding a mutant polypeptide having malate dehydrogenase activity as detailed herein above. Said MDH mutant may be a mutant of a homologous or heterologous wild-type MDH polypeptide. In a preferred embodiment, said MDH mutant is a mutant of a homologous MDH polypeptide. In an even more preferred embodiment, the recombinant yeast cell is a recombinant Saccharomyces, for example S. cerevisiae, and the MDH mutant is a mutant of a homologous MDH, for example MDH2 or MDH3. In a more specific embodiment, said recombinant yeast cell comprises a nucleic sequence encoding a mutant polypeptide having malate dehydrogenase activity as defined in Table 1 and wherein the amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39 is selected from glycine (G) or serine (S).

Standard genetic techniques, such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3^rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, N Y, 2001) or Ausubel et al. (Current protocols in molecular biology, Green Publishing and Wiley Interscience, N Y, 1987). Methods for transformation, genetic modification of fungal host cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671, WO90/14423, EP-A-0481008, EP-A-0635 574 and U.S. Pat. No. 6,265,186.

As used herein, the terms “nucleic acid”, “polynucleotide” or “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

The term “nucleic acid construct” is herein referred to as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally-occurring gene or, more typically, which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains all the control sequences required for expression of a coding sequence in a host cell, wherein said control sequences are operably linked to said coding sequence.

As used herein, the term “operably linked” refers to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

As used herein, the term “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences known to one of skilled in the art. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation.

A promoter that could be used to achieve the expression of a nucleotide sequence coding for an enzyme such a malate dehydrogenase or any other enzyme introduced in the host cell of the invention, may be not native to a nucleotide sequence coding for the enzyme to be expressed, i.e. a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operably linked. Preferably, the promoter is homologous, i.e. endogenous to the host cell.

Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art. Suitable promoters in eukaryotic host cells may be GAL7, GAL10, or GAL 1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, and AOX1. Other suitable promoters include PDC, GPD1, PGK1, TEF1, and TDH.

Usually a nucleotide sequence encoding an enzyme comprises a “terminator”. Any terminator, which is functional in the eukaryotic cell, may be used in the present invention. Preferred terminators are obtained from natural genes of the host cell. Suitable terminator sequences are well known in the art. Preferably, such terminators are combined with mutations that prevent nonsense mediated mRNA decay in the host cell of the invention (see for example: Shirley et al., 2002, Genetics 161:1465-1482).

The nucleic acid construct may be incorporated into a “vector”, such as an expression vector and/or into a host cell in order to effect expression of the polypeptide to be expressed.

The expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide encoding the polypeptide having malate dehydrogenase activity. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i. e., a vector, which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. If intended for use in a host cell of fungal origin, a suitable episomal nucleic acid construct may e.g. be based on the yeast 2p or pKD1 plasmids (Gleer et al., 1991, Biotechnology 9: 968-975), or the AMA plasmids (Fierro et al., 1995, Curr Genet. 29:482-489).

Alternatively, the expression vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The integrative cloning vector may integrate at random or at a predetermined target locus in the chromosomes of the host cell. In a preferred embodiment of the invention, the integrative cloning vector comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of host cell for targeting the integration of the cloning vector to this predetermined locus. In order to promote targeted integration, the cloning vector is preferably linearized prior to transformation of the cell. Linearization is preferably performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus. The length of the homologous sequences flanking the target locus is preferably at least 20 bp, at least 30 bp, at least 50 bp, at least 0.1 kb, at least 0.2 kb, at least 0.5 kb, at least 1 kb, at least 2 kb or longer. The efficiency of targeted integration into the genome of the host cell, i.e. integration in a predetermined target locus, is increased by augmented homologous recombination abilities of the host cell.

The homologous flanking DNA sequences in the cloning vector, which are homologous to the target locus, are derived from a highly expressed locus meaning that they are derived from a gene, which is capable of high expression level in the host cell. A gene capable of high expression level, i.e. a highly expressed gene, is herein defined as a gene whose mRNA can make up at least 0.5% (w/w) of the total cellular mRNA, e.g. under induced conditions, or alternatively, a gene whose gene product can make up at least 1% (w/w) of the total cellular protein, or, in case of a secreted gene product, can be secreted to a level of at least 0.1 g/l.

A nucleic acid construct or expression vector may be assembled in vivo in a host cell of the invention and, optionally, integrated into the genome of the cell in a single step (see, for example, WO2013/076280)

More than one copy of a nucleic acid construct or expression vector of the invention may be inserted into the host cell to increase production of the polypeptide having malate dehydrogenase activity (over-expression) encoded by the nucleic acid sequence comprised within the nucleic acid construct. This can be done, preferably by integrating into its genome two or more copies of the nucleic acid, more preferably by targeting the integration of the nucleic acid at a highly expressed locus defined as defined above.

It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.

A nucleic acid construct and/or expression vector of the invention can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell well known to those skilled in the art. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, N Y, 2001), Davis et al. (Basic Methods in Molecular Biology, 1^st edition, Elsevier, 1986) and other laboratory manuals.

Cytosolic expression of the enzymes described above may be obtained by deletion of a peroxisomal or mitochondrial targeting signal. The presence of a peroxisomal or mitochondrial targeting signal may for instance be determined by the method disclosed by Schlüter et al. (Schluter et al., 2007, Nucleic Acid Research 35: D815-D822).

A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp 276-277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as “longest-identity”.

The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the blastn and blastx programs (version 2.2.31 or above) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the blastn program, score=100, word-size=11 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the blastx program, score=50, word-size=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., blastx and blastn) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

According to the present invention, there is also provided a process for the production of a dicarboxylic acid, such as succinic acid, which process comprises fermenting the recombinant host cell of the invention as described herein above, under conditions suitable for production of the dicarboxylic acid, and optionally, recovering the dicarboxylic acid from the fermentation medium.

In the process, the recombinant host cell of the invention is fermented in a vessel comprising a suitable fermentation medium. The term fermenting, fermentation or fermented and the like as used herein refers to the microbial production of compounds, here dicarboxylic acids from carbohydrates.

Preferably, the fermentation product is a dicarboxylic acid, preferably malic acid, fumaric acid and/or succinic acid, preferably succinic acid.

A batch fermentation is defined herein as a fermentation wherein all nutrients are added at the start of a fermentation.

A fed-batch fermentation is a batch fermentation wherein the nutrients are added during the fermentation. Products in a batch and fed-batch fermentation may be harvested at a suitable moment, for instance when one or more nutrients are exhausted.

A continuous fermentation is a fermentation wherein nutrients are continuously added to the fermentation and wherein products are continuously removed from the fermentation.

In one embodiment fermenting the host cell in the process of the invention is carried out under carbohydrate limiting conditions. As used herein, carbohydrate limiting conditions are defined as maintaining the carbohydrate concentration below 10 g/l, for example about 5 g/l.

The process for the production of dicarboxylic acid according to the present invention may be carried out in any suitable volume and scale, preferably on an industrial scale. Industrial scale is defined herein as a volume of at least 10, or 100 litres, preferably at least 1 cubic metre, preferably at least 10, or 100 cubic metres, preferably at least 1000 cubic metres, usually below 10,000 cubic metres.

Fermenting the recombinant host cell in the process of the invention may be carried out in any suitable fermentation medium comprising a suitable nitrogen source, carbohydrate and other nutrients required for growth and production of a dicarboxylic acid in the process of the invention. A suitable carbohydrate in the fermentation process according to the invention may be glucose, galactose, xylose, arabinose, sucrose, or maltose.

In one embodiment, the fermentation process is carried out under a partial CO₂ pressure of between 5% and 60%, preferably about 50%.

The pH during the process for the production of dicarboxylic acid usually lowers during the production of the dicarboxylic acid. Preferably, the pH in the process for the production of dicarboxylic acid ranges between 1 and 5, preferably between 1.5 and 4.5, more preferably between 2 and 4.

In another preferred embodiment, the process according to the present invention comprises a step of preculturing the host cell under aerobic conditions in the presence of a carbohydrate. Preferably, the fermentation of the host cell during preculturing is carried out at a pH of between 4 and 6. Preferably, the carbohydrate during preculturing is a non-repressing carbohydrate, preferably galactose. It has been found advantageous to preculture host cells on a non-repressing carbohydrate, since this prevents glucose repression occurring, which may negatively influence the amount of biomass produced. In addition, it has been found that a step of preculturing host cells under aerobic conditions results in a higher biomass yield and a faster growth. Preferably, the preculturing is carried out in batch mode.

A propagation step for producing increased biomass is typically carried out, preferably under carbohydrate limiting conditions.

The process for producing a dicarboxylic acid may be carried out at any suitable temperature. A suitable temperature may for instance be between about 10 and about 40 degrees Celsius, for instance between about 15 and about 30 degrees Celsius.

In an embodiment, the process of the invention is carried out in such a way that at least a portion of the host cells is reused, i.e. recycled. The cells may be recycled back into the original vessel or into a second vessel. Preferably, the medium into which the recycled host cells are introduced is supplemented with a vitamin and/or a trace element.

In a preferred embodiment, the fermentation medium comprises an amount of succinic acid of between 1 and 150 g/l, preferably between 5 and 100 g/l, more preferably between 10 and 80 g/l or between 15 and 60 g/l of succinic acid. In any event, the recombinant host cell of the invention will typically be capable of accumulating more succinic acid in the fermentation medium as compared to a host cell that has been modified with a reference MDH polypeptide, for example that of SEQ ID NO: 39.

The process for the production of a dicarboxylic acid may further comprise recovering the dicarboxylic acid. Recovery of the dicarboxylic acid may be carried out by any suitable method known in the art, for instance by crystallization, ammonium precipitation, ion exchange technology, centrifugation or filtration or any suitable combination of these methods.

In a preferred embodiment, the recovery of the dicarboxylic acid comprises crystallizing the dicarboxylic acid and forming dicarboxylic acid crystals. Preferably, the crystallizing of the dicarboxylic acid comprises removing part of the fermentation medium, preferably by evaporation, to obtain a concentrated medium.

According to the present invention, the dicarboxylic acid, such as succinic acid may be recovered by crystallizing the dicarboxylic acid, such as succinic acid, from an aqueous solution having a pH of between 1 and 5 and comprising succinic acid, comprising evaporating part of the aqueous solution to obtain a concentrated solution, lowering the temperature of the concentrated solution to a value of between 5 and 35 degrees Celsius, wherein succinic acid crystals are formed. Preferably, the crystallizing comprises bringing the temperature of the concentrated medium to a temperature of between 10 and 30 degrees Celsius, preferably between 15 and 25 degrees Celsius. Preferably, the fermentation medium has a pH of between 1.5 and 4.5, preferably between 2 and 4.

It has been found that crystallizing the dicarboxylic acid, such as succinic acid, at higher temperatures such as between 10 and 30 degrees Celsius results in crystals of a dicarboxylic acid, such as succinic acid, with a lower amount of impurities such as organic acid, protein, color and/or odor, than crystals of a dicarboxylic acid, such as succinic acid, that were crystallized at a low temperature of below 10 degrees.

Another advantage of crystallizing succinic acid at a higher temperature is that it requires a lower amount of energy for cooling the aqueous solution as compared to a process wherein crystallizing the dicarboxylic acid is carried out below 10 or 5 degrees Celsius, resulting in a more economical and sustainable process.

Preferably, the crystallizing of the dicarboxylic acid, such as succinic acid, comprises a step of washing the dicarboxylic acid crystals. Dicarboxylic acid, such as succinic acid, may be crystallized directly from the fermentation medium having a pH of between 1 and 5 to a purity of at least 90% w/w, preferably at least 95, 96, 97, or at least 98%, or 99 to 100% w/w.

In a preferred embodiment, the process for the production of a dicarboxylic acid further comprises using the dicarboxylic acid in an industrial process.

Preferably, the dicarboxylic acid, such as succinic acid, that is prepared in the process according to the present invention is further converted into a desirable product. A desirable product may for instance be a polymer, such as polybutylene succinic acid (PBS), a deicing agent, a food additive, a cosmetic additive or a surfactant. That is to say, the invention provides a method for the production of a product, for example, a polymer, such as polybutylene succinic acid (PBS), a deicing agent, a food additive, a cosmetic additive or a surfactant, which method comprises: producing a dicarboxylic acid as described herein; and using said dicarboxylic acid in the production of said product.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

The present invention is further illustrated by the following Examples:

EXAMPLES

General Materials and Methods

DNA Procedures

Standard DNA procedures were carried out as described elsewhere (Sambrook et al., 1989, Molecular cloning: a laboratory manual, 2^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) unless otherwise stated. DNA was amplified using the proofreading enzyme Phusion polymerase (New England Biolabs, USA) according to manufacturer's instructions. Restriction enzymes were from Invitrogen or New England Biolabs.

Microtiter Plate (MTP) Fermentation of Dicarboxylic Acid Production Strains

To determine dicarboxylic acid production, strains were grown in triplicate in micro titer plates in humidity shakers (Infors) for 3 days at 30 degrees at 550 rpm and 80% humidity. The medium was based on Verduyn medium (Verduyn C, Postma E, Scheffers W A, Van Dijken J P. Yeast, 1992 July; 8(7):501-517), but modifications in carbon and nitrogen source were made as described herein below.

MTP pre-culture medium composition
		Concentration
Raw material		(g/l)
Galactose	C6H12O6•H2O	40.0
Urea	(NH2)2CO	2.3
Potassium	KH2PO4	3.0
dihydrogen
phosphate
Magnesium sulphate	MgSO4•7H2O	0.5
Trace element		1
solutiona
Vitamin solutionb		1
Component	Formula	Concentration (g/kg)
EDTA	C10H14N2Na2O8•2H2O	15.00
Zincsulphate•7H2O	ZnSO4•7H2O	4.50
Manganese-	MnCl2•2H2O	0.84
chloride•2H2O
Cobalt (II)	CoCl2•6H2O	0.30
chloride•6H2O
Copper (II)	CuSO4•5H2O	0.30
sulphate•5H2O
Sodium	Na2MoO4•2H2O	0.40
molybdenum•2H2O
Calcium-	CaCl2•2H2O	4.50
chloride•2H2O
Ironsulphate•7H2O	FeSO4•7H2O	3.00
Boric acid	H3BO3	1.00
Potassium iodide	Kl	0.10
Biotin (D−)	C10H16N2O3S	0.05
Ca D(+)	C18H32CaN2O10	1.00
panthothenate
Nicotinic acid	C6H5NO2	1.00
Myo-inositol	C6H12O6	25.00
Thiamine	C12H18Cl12N4OS•xH2O	1.00
chloride
hydrochloride
Pyridoxol	C8H12CINO3	1.00
hydrochloride
p-aminobenzoic acid	C7H7NO2	0.20
aTrace elements solution
bVitamin solution

80 microliters of pre-culture was used to inoculate 2.5 ml of medium with 1.5% galactose as carbon source in 24-well plates. The cultures were grown for 72 hours in humidity shakers (Infors) at 30° C., 550 rpm, 80% humidity. After generating biomass, a production experiment was started by re-suspending cells into 2.5 ml of mineral medium with glucose as carbon source. The main cultures were incubated in humidity shakers (Infors) at 30 degrees at 550 rpm and 80% humidity and samples were taken after 48 hours of cultivation.

Metabolite Analysis of MTP Samples by NMR

For metabolite analysis of MTP samples, 90 microliter of supernatant of fermentation samples is mixed with 10 microliter of NMR standard (20 g/l maleic acid) and 100 microliter of 10% D₂O solution. The samples are lyophilized and subsequently dissolved in 1 mL D₂O.

1D 1H NMR spectra are recorded on a BEST Bruker Avance III spectrometer, operating at a proton frequency of 500 MHz, equipped with a He-cooled cryo probe, using a pulse program without water suppression (ZG) at a temperature of 300 K, with a 90 degree excitation pulse, acquisition time of 2.0 seconds and a relaxation delay of 40 seconds. The number of scans was set at 8.

The malic acid concentration [in g/l] is calculated based on the following signals (δ relative to 4,4-dimethyl-4-silapentane-1-sulfonic acid):

Malic acid: Depending on the pH and overlap of the α-CH2 and the CH(OH) signals with other compounds, one of the three malic acid signals is chosen for quantification, α-CH(A) (2.92 ppm, n=1H, double doublet or dd), α-CH(X) (2.85 ppm, n=1H, dd) or CH(OH) (4.6 ppm, n=1H, dd).

The succinic acid concentration [in g/L] is calculated based on the following signals (δ relative to 4,4-dimethyl-4-silapentane-1-sulfonic acid):

Succinic acid: succinic acid signal at 2.67 ppm (s, 4H)

The signal used for the standard: maleic acid peak around 6.3 ppm (S, 2H).

Quantification by NMR is described by Bharti et al., 2012, TrAC Trends in Analytical Chemistry 35:5-26.

Example 1: Construction of Strain SUC-1029

Strain CEN.PK 113-7D (MATa HIS3 LEU2 TRP1 MAL2-8 SUC2) was used as a starting point to construct strain SUC-1029. A fumarase gene of Rhyzopus oryzae (FUMR) was transformed to strain CEN.PK113-7D as described below.

Generation of PCR Fragments

PCR fragment 9 was obtained by PCR amplification of SEQ ID NO: 34 using primers amplifying the entire nucleotide sequence of SEQ ID NO: 34. SEQ ID NO: 34 describes a synthetic polynucleotide containing the fumarase (FUMR) nucleotide sequence from Rhyzopus oryzae as disclosed in patent application WO2009/065779. The gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632. Expression of the FUMR gene is controlled by the TDH1 promoter (600 bp directly before the start codon of the TDH1 gene) and the TDH1 terminator (300 bp directly after the stop codon of the TDH1 gene). The TDH1 promoter and TDH1 terminator sequences controlling expression of FUMR are native sequences derived from Saccharomyces cerevisiae S288C. The 599 bp region at the 5′ end of SEQ ID NO: 34, upstream of the TDH1 promoter, is a region homologous to the YPRCtau3 locus.

PCR fragment 10 was obtained by PCR amplification of SEQ ID NO: 35 using primers amplifying the entire nucleotide sequence of SEQ ID NO: 35. SEQ ID NO: 35 describes a synthetic polynucleotide containing part of the pSUC227 plasmid sequence, described in PCT/EP2013/055047. The 5′ end of SEQ ID NO: 35 contains overlap with the 3′ end of SEQ ID NO: 34. The 3′ end of SEQ ID NO: 35 contains overlap with the 5′ end of SEQ ID NO: 36.

PCR fragment 11 was obtained by PCR amplification of SEQ ID NO: 36 using primers amplifying the entire nucleotide sequence of SEQ ID NO: 36. SEQ ID NO: 36 describes a synthetic polynucleotide containing part of the pSUC225 plasmid sequence, described in PCT/EP2013/055047. The 3′ end of SEQ ID NO: 36 contains overlap with the 5′ end of SEQ ID NO: 37.

PCR fragment 12 was obtained by PCR amplification of SEQ ID NO: 37 using primers amplifying the entire nucleotide sequence of SEQ ID NO: 37. SEQ ID NO: 37 describes a synthetic polynucleotide homologous to the YPRCtau3 locus.

PCR fragments 9 to 12 were purified using the DNA Clean & Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA) according to manufacturer's instructions.

Transformation to CEN.PK113-7D in Order to Construct Strain SUC-1029

Yeast transformation was done by a method known by persons skilled in the art. S. cerevisiae strain CEN.PK113-7D was transformed with purified PCR fragments 9 to 12 PCR fragments 10 and 11 contained overlaps at their 5′ and 3′ ends and PCR fragments 9 and 12 at their 3′ and 5′ end respectively, such that this allowed homologous recombination of all four PCR fragments (FIG. 1). The 5′ end of PCR fragment 9 and the 3′ end of PCR fragment 12 were homologous to the YPRCtau3 locus and enabled integration of all four PCR fragments in the YPRCtau3 locus (FIG. 1). This resulted in one linear fragment consisting of PCR fragments 9 to 12 integrated in the YPRCtau3 locus, which is located on chromosome XVI.

Transformation mixtures were plated on YEPhD-agar (per liter: 10 grams yeast extract, 20 grams PhytonePeptone, 20 grams glucose, 20 grams agar) containing 200 μg G418 (Sigma Aldrich, Zwijndrecht, The Netherlands) per ml. After three days of growth at 30° C., individual transformants were re-streaked on YEPh-agar plates containing 20 grams glucose per liter and 200 μg G418 per ml.

Subsequently, the marker cassette and Cre-recombinase gene present on the integrated PCR fragments 10 and 11 were removed by recombination between the lox66 and lox71 sites that flank the KanMX marker and the CRE gene encoding the CRE recombinase by CRE recombinase, using the method described in PCT/EP2013/055047, resulting in removal of the KanMX marker and the CRE gene and leaving a lox72 site as a result of recombination between the lox66 and lox71 sites. The resulting markerfree strain was named SUC-1029.

Presence of the introduced FUMR gene was confirmed by using PCR using primer sequences that can anneal to the coding sequences of the ORF's encoded by SEQ ID NO: 34. Correct integration and removal of the KanMX marker was confirmed by PCR using primers 5′ and 3′ from the YPRCtau3 locus, not hybridizing on the YPRCtau3 homologous regions present on PCR fragments 9 and 12.

Example 2: Construction of Strain SUC-1112

Generation of PCR Fragments

Primer sequences described in SEQ ID NO: 9 and SEQ ID NO: 10 were used to generate PCR fragment 1 consisting of the 5′ INT59 integration site, using genomic DNA of strain Saccharomyces cerevisiae strain CEN.PK 113-7D (MATa HIS3 LEU2 TRP1 MAL2-8 SUC2) as template.

PCR fragment 2 was generated by using the primer sequences described in SEQ ID NO: 11 and SEQ ID NO: 12, using SEQ ID NO: 1 as template. SEQ ID NO: 1 encodes phosphoenolpyruvate carboxykinase (PCKa) from Actinobacillus succinogenes, as disclosed in patent application WO2009/065780. This synthetic sequence, which includes promoter-gene-terminator sequence, including appropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). The gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632. The synthetic gene is under control of (or operable linked to) a promoter from S. cerevisiae, i.e. the TPI1-promoter controls the expression of the PCKa-gene. Proper termination is controlled by a terminator sequence from S. cerevisiae, i.e. the GND2-terminator.

PCR fragment 3 was generated by using the primer sequences described in SEQ ID NO: 13 and SEQ ID NO: 14, using SEQ ID NO: 2 as template. SEQ ID NO: 2 encodes pyruvate carboxylase (PYC2) from Saccharomyces cerevisiae, as disclosed in patent application WO2009/065780. This synthetic sequence, which includes promoter-gene-terminator sequence, including appropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). The gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632. The synthetic gene is under control of (or operable linked to) a promoter from S. cerevisiae, i.e. the PGK1-promoter controls the expression of the PYC2-gene. Proper termination is controlled by a terminator sequence from S. cerevisiae, i.e. the ADH1-terminator.

PCR fragment 4 was generated by using the primer sequences described in SEQ ID NO: 15 and SEQ ID NO: 16, using SEQ ID NO: 3 as template. SEQ ID NO: 3 encodes a KanMX selection marker functional in Saccharomyces cerevisiae which was amplified from plasmid pUG7-EcoRV. pUG7-EcoRV is a variant of plasmid pUG6 described by Gueldener et al., (Nucleic Acids Res. 1996 Jul. 1; 24(13):2519-24), in which the loxP sites present in pUG6 were changed into lox66 and lox71 sites (Lambert et al., Appl. Environ. Microbiol. 2007 February; 73(4):1126-35. Epub 2006 Dec. 1.)

PCR fragment 5 was generated by using the primer sequences described in SEQ ID NO: 17 and SEQ ID NO: 18, using SEQ ID NO: 4 as template. SEQ ID NO: 4 encodes a putative dicarboxylic acid transporter from Aspergillus niger, as disclosed in EP2495304. This synthetic sequence, which includes promoter-gene-terminator sequence, including appropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). The gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632. The synthetic gene is under control of (or operable linked to) a promoter from S. cerevisiae, i.e. the ENO1-promoter controls the expression of the DCT_02-gene. Proper termination is controlled by a terminator sequence from S. cerevisiae, i.e. the TEF2-terminator.

PCR fragment 6 was generated by using the primer sequences described in SEQ ID NO: 19 and SEQ ID NO: 20, using SEQ ID NO: 5 as template. SEQ ID NO: 5 encodes malate dehydrogenase (MDH3) from Saccharomyces cerevisiae, as disclosed in patent application WO2009/065778. This synthetic sequence, which includes promoter-gene-terminator sequence, including appropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). The gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632. The synthetic gene is under control of (or operable linked to) a promoter from Kluyveromyces lactis, i.e. the promoter of ORF KLLA0_F20031g (uniprot accession number Q6CJA9) controls the expression of the MDH3-gene. Proper termination is controlled by a terminator sequence from S. cerevisiae, i.e. the GPM1-terminator.

PCR fragment 7 was generated by using the primer sequences described in SEQ ID NO: 21 and SEQ ID NO: 22, using SEQ ID NO: 6 as template. SEQ ID NO: 6 encodes fumarase (fumB) from Escherichia coli (E.C. 4.2.1.2, UniProt accession number P14407). The gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632. The synthetic sequence, which includes promoter-gene-terminator sequence, including appropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). The synthetic gene is under control of (or operable linked to) a promoter from S. cerevisiae, i.e. the TDH3-promoter controls the expression of the controls the expression of the fumB-gene. Proper termination is controlled by a terminator sequence from S. cerevisiae, i.e. the TDH1-terminator.

PCR fragment 8 was generated by using the primer sequences described in SEQ ID NO: 23 and SEQ ID NO: 24, using SEQ ID NO: 7 as template. SEQ ID NO: 7 encodes encodes fumarate reductase (FRDg) from Trypanosoma brucei, as disclosed in patent application WO2009/065778. The gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632. The synthetic sequence, which includes promoter-gene-terminator sequence, including appropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). The synthetic gene is under control of (or operable linked to) a promoter from S. cerevisiae, i.e. the TEF1-promoter controls the expression of the controls the expression of the fumB-gene. Proper termination is controlled by a terminator sequence from S. cerevisiae, i.e. the TAL1-terminator.

Primer sequences described in SEQ ID NO: 40 and SEQ ID NO: 41 were used to generate PCR fragment 113 consisting of the 3′ INT59 integration site, using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragments 1 to 8 and PCR fragment 113 were purified using the DNA Clean & Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA) according to manufacturer's instructions.

Transformation to SUC-1029 in Order to Construct Strain SUC-1112

Yeast transformation was done by a method known by persons skilled in the art. S. cerevisiae strain SUC-1029 was transformed with purified PCR fragments 1 to 8 and PCR fragment 113. PCR fragments 2 to 8 contained overlaps at their 5′ and 3′ ends and PCR fragments 1 and 113 at their 3′ and 5′ end respectively, such that this allowed homologous recombination of all eight PCR fragments. The 5′ end of PCR fragment 1 and the 3′ end of PCR fragment 113 were homologous to the INT59 locus and enabled integration of all nine PCR fragments in the INT59 locus (see FIG. 2). This resulted in one linear fragment consisting of PCR fragments 2 to 8 integrated in the INT59 locus. This method of integration is described in patent application WO2013076280. The INT59 locus is located at chromosome XI, 923 bp downstream of YKR092C and 922 bp upstream of YKR093W.

Transformation mixtures were plated on YEPh-agar (per liter: 10 grams yeast extract, 20 grams PhytonePeptone, 20 grams agar) containing 20 grams galactose per liter and 200 μg G418 (Sigma Aldrich, Zwijndrecht, The Netherlands) per ml. After three days of growth at 30° C., individual transformants were re-streaked on YEPh-agar plates containing 20 grams galactose per liter and 200 μg G418 per ml. Presence of all introduced genes was confirmed by using PCR using primer sequences that can anneal to the coding sequences of the ORF's encoded by SEQ ID NO: 1 to SEQ ID NO: 7. The resulting strain was named SUC-1112. The KanMX marker, present in strain SUC-1112, can be removed if required.

Example 3: Transformation of a Malate Dehydrogenase Gene to Strain SUC-1112 and Production of Malic Acid in Resulting Transformants

Generation of PCR Fragments

Primer sequences described in SEQ ID NO: 25 and SEQ ID NO: 26 were used to generate PCR fragment 13 consisting of the 5′ INT1 integration site, using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragment 114 was generated by using the primer sequences described in SEQ ID NO: 43 and SEQ ID NO: 44, using SEQ ID NO: 42 as template. SEQ ID NO: 42 contains the ZWF1 gene, encoding Glucose-6-phosphate dehydrogenase (G6PD). This synthetic sequence, which includes promoter-gene-terminator sequence, including appropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). The gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632. The synthetic gene is under control of (or operable linked to) a promoter from Kluyveromyces lactis, i.e. the promoter of ORF KLLA0C05566g (uniprot accession number Q6CUE2) controls the expression of the ZWF1-gene. Proper termination is controlled by a terminator sequence from S. cerevisiae, i.e. the TEF1-terminator.

PCR fragment 115 was generated by using the primer sequences described in SEQ ID NO: 45 and SEQ ID NO: 28, using SEQ ID NO: 38 as template. SEQ ID NO: 38 encodes a nourseothricin selection marker functional in Saccharomyces cerevisiae which was amplified from a modified version of plasmid pUG7-Nat. pUG7-Nat is a variant of plasmid pUG6 described by Gueldener et al., (Nucleic Acids Res. 1996 Jul. 1; 24(13):2519-24), in which the loxP sites present in pUG6 were changed into lox66 and lox71 sites (Lambert et al., Appl. Environ. Microbiol. 2007 February; 73(4):1126-35. Epub 2006 Dec. 1) and in which the KanMX marker was replaced by a nourseothricin marker (Goldstein and McCusker, Yeast. 1999 October; 15(14):1541-53).

PCR fragment 15 was generated by using the primer sequences described in SEQ ID NO: 29 and SEQ ID NO: 30, using SEQ ID NO: 31 as template. SEQ ID NO: 31 encodes malate dehydrogenase (MDH3) from S. cerevisiae. MDH3 is altered by the deletion of the SKL carboxy-terminal sequence as disclosed in patent application WO2013/004670 A1. This synthetic sequence, which includes promoter-gene-terminator sequence, including appropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). The gene sequence was codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632. The synthetic gene is under control of (or operable linked to) a promoter from S. cerevisiae, i.e. the TDH3-promoter controls the expression of the MDH3-gene. Proper termination is controlled by a terminator sequence from S. cerevisiae, i.e. the GPM1-terminator.

Primer sequences described in SEQ ID NO: 32 and SEQ ID NO: 33 were used to generate PCR fragment 16 consisting of the 3′ INT1 integration site, using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragments 13 to 16 were purified using the DNA Clean & Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA) according to manufacturer's instructions.

Transformation to SUC-1112

Yeast transformation was done by a method known by persons skilled in the art. S. cerevisiae strain SUC-1112 was transformed with purified PCR fragments 13, 114, 115, 15 and 16. PCR fragments 114 and 115 and 15 contained overlaps at their 5′ and 3′ ends and PCR fragments 13 and 16 contained overlaps at their 3′ and 5′ end respectively, such that this allowed homologous recombination of all five PCR fragments. The 5′ end of PCR fragment 13 and the 3′ end of PCR fragment 16 were homologous to the INT1 locus and enabled integration of all four PCR fragments in the INT1 locus (see FIG. 3). This resulted in one linear fragment consisting of PCR fragments 13 to 16 integrated in the INT1 locus. This method of integration is described in patent application WO2013/076280. The INT1 locus is located at chromosome XV, 659 bp downstream of YOR071c and 998 bp upstream of YOR070c. This approach resulted in expression of the malate dehydrogenase protein of 340 amino acids as indicated in SEQ ID NO: 39 which lacks the C-terminal amino acid SKL as compared to the native sequence from S. cerevisiae.

Transformation mixtures were plated on YEPh-agar (per liter: 10 grams yeast extract, 20 grams PhytonePeptone, 20 grams agar)) containing 20 grams galactose per liter and 200 μg nourseothricin (Jena Bioscience, Germany) per ml. After three days of growth at 30° C., individual transformants were re-streaked on YEPh-agar plates containing 20 grams galactose per liter and 200 μg nourseothricin per ml. Presence of the introduced genes was confirmed by using PCR using primer sequences that can anneal to the coding sequences of the ORF's encoded present on PCR fragment 114, 115 and 15. To confirm integration of PCR fragments 13, 114, 115, 15 and 16 on the correct locus, primers annealing to the region 5′ and 3′ of the INT1 locus, not binding to the INT1 regions on PCR fragments 13 and 16 were used in combination with primers annealing to the ORF's on PCR fragments 114 and 15 such that only PCR product can be formed if PCR fragments 114 and 15 are integrated in the INT1 locus. Three resulting individual colonies SUC-1112+MDH3 #1, SUC-1112+MDH3 #2, SUC-1112+MDH3 #3. The KanMX and nourseothricin markers, present in strains SUC-1112+MDH3 #1, SUC-1112+MDH3 #2, SUC-1112+MDH3 #can be removed if required.

Dicarboxylic Acid Production

To determine dicarboxylic acid production MTP fermentations and NMR measurements were performed as described in General Materials and Methods.

In the supernatant of the SUC-1112+MDH3 strains, SUC-1112+MDH3 #1, SUC-1112+MDH3 #2, SUC-1112+MDH3 #3, which contain an additional copy of the MDH3 gene, present on PCR fragment 15, an average titer of 8.7 g/L malic acid was measured. Succinic acid levels were lower than expected; the strains appeared to have lost the FRDg gene resulting in limited conversion of malate to succinate.

Example 4: Transformation of Genes Encoding Malate Dehydrogenase Mutants to Strain SUC-1112 and Production of Malic Acid in Resulting Transformants

Generation of PCR fragments

PCR fragments 13 and 16 were generated as described in Example 3.

PCR fragment 14 was generated by using the primer sequences described in SEQ ID NO: 27 and SEQ ID NO: 28, using SEQ ID NO: 38 as template. SEQ ID NO: 38 encodes a nourseothricin selection marker functional in Saccharomyces cerevisiae which was amplified from a modified version of plasmid pUG7-Nat. pUG7-Nat is a variant of plasmid pUG6 described by Gueldener et al., (Nucleic Acids Res. 1996 Jul. 1; 24(13):2519-24), in which the loxP sites present in pUG6 were changed into lox66 and lox71 sites (Lambert et al., Appl. Environ. Microbiol. 2007 February; 73(4):1126-35. Epub 2006 Dec. 1) and in which the KanMX marker was replaced by a nourseothricin marker (Goldstein and McCusker, Yeast. 1999 October; 15(14):1541-53).

Synthetic nucleotide sequences encoding different protein mutants of the reference malate dehydrogenase sequence that is described in SEQ ID NO: 39 were synthesized by DNA 2.0 (Menlo Park, Calif., USA). The synthetic nucleotide sequences encode a mutant amino acid sequence at positions 34 to 40 relative to the reference MDH3 sequence (SEQ ID NO: 39) as indicated in Table 1. Apart from encoding the indicated mutant amino acids in Table 1 the synthetic nucleotide sequence mutants are identical to SEQ ID NO: 31 The synthetic gene is under control of (or operable linked to) a promoter from S. cerevisiae, i.e. the TDH3-promoter controls the expression of the mutant MDH-gene. Proper termination is controlled by a terminator sequence from S. cerevisiae, i.e. the GPM1-terminator.

The synthetic gene sequences containing amongst others a TDH3 promoter-mutant MDH-GPM1 terminator and were amplified by PCR using the primer sequences described in SEQ ID NO: 29 and SEQ ID NO: 30, to generate PCR fragments 17 to 108 (see Table 1).

PCR fragments 13, 14, 16 and 17 to 108 were purified using the DNA Clean & Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA) according to manufacturer's instructions.

Transformation to SUC-1112

Strain SUC-1112 was transformed with purified PCR fragments 13, 14 and 16 in combination with PCR fragments 17 to 108 individually. PCR fragment 14 and PCR fragments 17 to 108 contained overlaps at their 5′ and 3′ ends and PCR fragments 13 and 16 contained overlaps at their 3′ and 5′ end respectively, such that this allowed homologous recombination of all four PCR fragments. The 5′ end of PCR fragment 13 and the 3′ end of PCR fragment 16 were homologous to the INT1 locus and enabled integration of all four PCR fragments in the INT1 locus (FIG. 4). Transformation and selection of transformants is described in Example 2.

Dicarboxylic Acid Production

To determine dicarboxylic acid production, four independent SUC-1112 transformants expressing mutant malate dehydrogenase sequences were grown in micro titer plates as described in General Materials and Methods. Low amounts of succinic acid were produced due to the loss of FRDg (see Example 3), but MDH3 activity could still be determined by measuring malate levels. Average malic acid titers are depicted in Table 1. The average production of malic acid of several SUC-1112 transformants expressing mutant malate dehydrogenase sequences exceeded 10 g/L malic acid. Interestingly, mutants with a substitution of aspartic acid by a glycine or serine residue at position 34 show increased malate production. This is significantly more than the average malic acid titer of SUC-1112 transformed with the reference MDH3 sequence described in Example 3. By significantly more it is meant that the 95% confidence intervals of malic titers for strains with reference and improved mutant malate dehydrogenase sequences do not overlap. The upper limit of the 95% confidence interval for the malic acid titer of SUC-1112 transformed with the reference MDH3 sequence lies below 10 g/L.

TABLE 1
Average malic acid titers measured in the supernatant of production
medium after 4 days cultivation of transformants of strain
SUC-1112, expressing phosphoenolpyruvate carboxykinase (PCKa),
pyruvate carboxylase (PYC2), malate dehydrogenase (MDH3),
fumarase (FUMR and fumB), dicarboxylic acid transporter
(DCT 02), and transformed with the nucleotide sequence
encoding the reference malate dehydrogenase (SEQ ID NO: 39)
or a malate dehydrogenase mutant (MUT 001-MUT 94), which
contains mutations as compared to the reference sequence in the
amino acid positions indicated below.
PCR		Loop sequence	Average
frag-		(amino acid position)	malic acid
ment	Clone	34	35	36	37	38	39	40	titer (g/L)
15	SUC-1112 +	D	I	R	A	A	E	G	8.7 g/L
	MDH3 reference
17	MUT_001	D	I	Q	A	A	E	G	9.0
18	MUT_002	D	I	S	A	A	E	G	9.1
19	MUT_003	D	I	R	A	A	E	G	9.0
20	MUT_004	D	I	Q	A	A	E	G	9.7
21	MUT_005	D	S	S	A	A	E	G	9.8
22	MUT_006	S	I	R	A	A	E	G	10.1
23	MUT_007	S	I	Q	A	A	E	G	11.7
24	MUT_008	S	I	S	A	A	E	G	17.1
25	MUT_009	S	S	R	A	A	E	G	16.1
26	MUT_010	S	S	Q	A	A	E	G	16.4
27	MUT_011	S	S	S	A	A	E	G	16.5
28	MUT_012	G	I	R	A	A	E	G	16.6
29	MUT_013	G	I	Q	A	A	E	G	16.2
30	MUT_014	G	I	S	A	A	E	G	16.5
31	MUT_015	G	S	R	A	A	E	G	15.8
32	MUT_016	G	S	Q	A	A	E	G	16.5
33	MUT_017	G	S	S	A	A	E	G	15.9
34	MUT_018	D	I	A	V	T	P	G	9.1
35	MUT_019	D	I	A	N	V	K	G	9.0
36	MUT_020	D	I	A	N	V	K	G	9.2
37	MUT_021	D	I	Q	N	V	K	G	9.2
38	MUT_022	D	I	S	N	V	K	G	8.8
39	MUT_023	D	S	A	N	V	K	G	9.3
40	MUT_024	D	S	R	N	V	K	G	9.3
41	MUT_025	D	S	Q	N	V	K	G	8.4
42	MUT_026	D	S	S	N	V	K	G	9.3
43	MUT_027	S	I	A	N	V	K	G	15.4
44	MUT_028	S	I	R	N	V	K	G	14.4
45	MUT_029	S	I	Q	N	V	K	G	11.7
46	MUT_030	S	I	S	N	V	K	G	16.1
47	MUT_031	S	S	A	N	V	K	G	15.8
48	MUT_032	S	S	R	N	V	K	G	15.8
49	MUT_033	S	S	Q	N	V	K	G	15.7
50	MUT_034	S	S	S	N	V	K	G	16.1
51	MUT_035	G	I	A	N	V	K	G	15.7
52	MUT_036	G	I	R	N	V	K	G	14.2
53	MUT_037	G	I	Q	N	V	K	G	9.5
54	MUT_038	G	I	S	N	V	K	G	16.6
55	MUT_039	G	S	A	N	V	K	G	11.8
56	MUT_040	G	S	R	N	V	K	G	14.4
57	MUT_041	G	S	Q	N	V	K	G	15.4
58	MUT_042	G	S	S	N	V	K	G	15.9
59	MUT_043	D	I	A	G	T	P	G	8.3
60	MUT_044	D	I	R	G	T	P	G	7.6
61	MUT_045	D	I	Q	G	T	P	G	7.9
62	MUT_046	D	I	S	G	T	P	G	8.5
63	MUT_047	D	S	A	G	T	P	G	8.4
64	MUT_048	D	S	R	G	T	P	G	8.4
65	MUT_049	D	S	Q	G	T	P	G	8.7
66	MUT_050	D	S	S	G	T	P	G	7.4
67	MUT_051	S	I	A	G	T	P	G	9.7
68	MUT_052	S	I	R	G	T	P	G	11.3
69	MUT_053	S	I	Q	G	T	P	G	13.8
70	MUT_054	S	I	S	G	T	P	G	13.4
71	MUT_055	S	S	A	G	T	P	G	14.4
72	MUT_056	S	S	R	G	T	P	G	13.9
73	MUT_057	S	S	Q	G	T	P	G	14.2
74	MUT_058	S	S	S	G	T	P	G	14.2
75	MUT_059	G	I	A	G	T	P	G	13.9
76	MUT_060	G	I	R	G	T	P	G	13.5
77	MUT_061	G	I	Q	G	T	P	G	13.7
78	MUT_062	G	I	S	G	T	P	G	14.2
79	MUT_063	G	S	A	G	T	P	G	14.7
80	MUT_064	G	S	R	G	T	P	G	11.8
81	MUT_065	G	S	Q	G	T	P	G	14.4
82	MUT_066	G	S	S	G	T	P	G	14.4
83	MUT_067	D	I	E	R	S	F	Q	6.5
84	MUT_068	D	I	E	R	S	F	G	6.4
85	MUT_069	D	I	E	A	S	F	Q	8.6
86	MUT_070	D	I	E	A	S	F	G	8.3
87	MUT_071	D	S	E	R	S	F	Q	7.6
88	MUT_072	D	S	E	R	S	F	G	8.5
89	MUT_073	D	S	E	A	S	F	Q	6.9
90	MUT_074	D	S	E	A	S	F	G	8.0
91	MUT_075	S	I	E	R	S	F	Q	9.7
92	MUT_076	S	I	E	R	S	F	G	15.1
93	MUT_077	S	I	E	A	S	F	Q	7.8
94	MUT_078	S	I	E	A	S	F	G	13.7
95	MUT_079	S	S	E	R	S	F	Q	14.1
96	MUT_080	S	S	E	R	S	F	G	14.1
97	MUT_081	S	S	E	A	S	F	Q	12.2
98	MUT_082	S	S	E	A	S	F	G	13.9
99	MUT_083	G	I	E	R	S	F	Q	9.3
100	MUT_084	G	I	E	R	S	F	G	14.0
101	MUT_085	G	I	E	A	S	F	Q	6.3
102	MUT_086	G	I	E	A	S	F	G	12.2
103	MUT_087	G	S	E	R	S	F	Q	14.0
104	MUT_088	G	S	E	R	S	F	G	15.9
105	MUT_089	G	S	E	A	S	F	Q	14.0
106	MUT_090	G	S	E	A	S	F	G	14.5
107	MUT_091	D	I	P	Q	A	L	G	7.6
108	MUT_092	D	S	P	Q	A	L	G	7.3
109	MUT_093	S	S	P	Q	A	L	G	14.2
110	MUT_094	G	S	P	Q	A	L	G	16.0

Example 5: Measuring NADH and NADPH Specific Activity of Malate Dehydrogenase Mutants

A total of 19 mutants were selected from Table 1 and re-cultured as described in General materials and methods. The biomass was harvested by centrifugation (4000 rpm, 10 min, 4° C.) and washed twice with PBS (phosphate buffered saline, Sigma Aldrich) after which the cell pellets were frozen at −20° C. Cell disruption was achieved in square welled 96-deepwell micro titer plates (MTP) using 0.5 mm acid washed glass beads in combination with the TissueLyser II from Qiagen (3000 rpm for 2×10 sec, cool on ice for 1 min between cycles). Glass beads taking up a volume of 600 μl were added to the cell pellet before addition of 1 ml in vivo like-assay medium (described in van Eunen et al. FEBS Journal 277: 749-760 adapted to contain 0.5 mM DTT (dithiothreitol, Sigma-Aldrich) and 0.1 mM PMSF (phenylmethanesulfonyl fluoride, Amresco). Glass beads were added by inverting the deep well MTP containing the frozen pellets over a standard MTP where each well is filled completely with glass beads (=a volume of 300 μl) and then inverting both plates, so that the glass beads fall onto the cell pellets. This process was repeated to obtain 600 μl glass beads in the cell pellets. Next 1 ml of in vivo like-assay medium described above was added. After cell disruption, cell debris was pelleted by centrifugation (4000 rpm, 30 min, 4° C.). The supernatant (soluble cell extracts) were collected and stored on ice. Protein concentration of the extracts was determined by Bradford, using bovine serum albumin (BSA) as standard.

Malate dehydrogenase (MDH) activity was assayed spectrophotometrically by following the decrease in absorbance at 340 nm caused by the oxidation of NADH or NADPH to NAD+ or NADP+, respectively. Assays contained 400 μM NADH or 400 μM NADPH, 2 mM oxaloacetic acid (Sigma Aldrich) and approximately 0.0625 mg protein ml⁻¹ soluble cell extracts in in vivo-like assay medium. Assays were performed in a final volume of 200 μl. Equal volume of soluble cell extracts were added in both the NADH and NADPH dependent assays. Reactions were started by the addition of 100 μl oxaloacetic acid stock solution (4 mM) and were followed for 9 minutes at 30 degrees Celsius and the slope was used as a measure of NADH or NADPH dependent MDH activity. The slope (in Δ A340/min) was determined with the ‘slope’ function in Microsoft Excel where the absorbance values were taken as ‘y’ values and the time in minutes as ‘x’ values. The ‘RSQ’ function in Microsoft Excel was used to check the quality of the slope fitting (criteria >0.9). The slope was corrected for the slope of blank reaction containing in vivo-like assay medium instead of substrate. Absorbance was measured using a Tecan Infinite M1000 plate reader. NADH dependent activity of each mutant was compared to the NADPH activity. The ratio of NADPH:NADH dependent activity or NADPH:NADH specificity ratio, was determined by:

• • 1) Determining the slope (in Δ A340/min) of the NADPH-dependent MDH activity
  • 2) Determining the slope (in Δ A340/min) NADH-dependent MDH activity
  • 3) Dividing the slope of the NADPH-dependent MDH activity by the slope of the NADH-dependent MDH activity.
  • To determine the ratio, slopes were used without normalization for amount of total protein as equal volumes of each mutant were used in the NADH and NADPH dependent MDH activity assays,
    The ratio was calculated for 19 mutants (FIG. 5D). An increased value for the ratio compared to the reference indicated that the cofactor specificity has been changed.

Supernatants of the 19 cultured mutants and the reference strain were analysed for malic acid titers as described in general materials and methods. The NADPH-specific and NADH-specific activities were measured as described above and normalized for total protein by dividing by the total protein concentration in the assay. The results are shown in FIGS. 5B-5C. Clearly, the 19 selected mutants have an enhanced NADPH-specific malate dehydrogenase activity. For most of the 19 mutants the NADH-specific malate dehydrogenase activity is decreased compared to the reference (FIG. 5B). Interestingly, in 6 mutants, the NADH-specific activity is increased (FIG. 5B), indicating that in these mutants both the NADH-specific activity and NADPH-specific activity is increased. In all mutants, the NADPH:NADH specificity ratio was increased (FIG. 5D).

Surprisingly, a substitution of aspartic acid by a glycine or serine residue at position 34 has a positive effect on the malic acid titer of SUC-1112 strains transformed with these malate dehydrogenase mutants (FIG. 5A).

Example 6: Construction of Strain REV-0001

Generation of PCR Fragments

Primer sequences described in SEQ ID NO: 54 and SEQ ID NO: 55 are used to generate PCR fragment 116 consisting of the 5′ INT1 integration site, using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragment 117 is generated by using the primer sequences described in SEQ ID NO: 56 and SEQ ID NO: 57, using SEQ ID NO: 38 as template. SEQ ID NO: 38 encodes a nourseothricin selection marker functional in Saccharomyces cerevisiae which was amplified from a modified version of plasmid pUG7-Nat. pUG7-Nat is a variant of plasmid pUG6 described by Gueldener et al., (Nucleic Acids Res. 1996 Jul. 1; 24(13):2519-24), in which the loxP sites present in pUG6 were changed into lox66 and lox71 sites (Lambert et al., Appl. Environ. Microbiol. 2007 February; 73(4):1126-35. Epub 2006 Dec. 1) and in which the KanMX marker was replaced by a nourseothricin marker (Goldstein and McCusker, Yeast. 1999 October; 15(14):1541-53).

PCR fragment 118 is generated by using the primer sequences described in SEQ ID NO: 60 and SEQ ID NO: 61, using SEQ ID NO: 62 as template. SEQ ID NO: 62 encodes fumarate reductase (FRDg) from Trypanosoma brucei, as disclosed in patent application WO2009/065778. This synthetic sequence, which includes promoter-gene-terminator sequence, including appropriate restriction sites, is synthesized by DNA 2.0 (Menlo Park, Calif., USA). The gene sequence is codon pair optimized for expression in S. cerevisiae as disclosed in patent application WO2008/000632. The synthetic gene is under control of (or operable linked to) a promoter from S. cerevisiae, i.e. the TDH3-promoter controls the expression of the FRDg-gene. Proper termination is controlled by a terminator sequence from S. cerevisiae, i.e. the TAL1-terminator. Primer sequences described in SEQ ID NO: 58 and SEQ ID NO: 59 are used to generate PCR fragment 119 consisting of the 3′ INT1 integration site, using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragments 116 to 119 are purified using the DNA Clean & Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA) according to manufacturer's instructions.

Transformation of SUC-1029

Yeast transformation is performed by a method known by persons skilled in the art. S. cerevisiae strain SUC-1029 (Example 1) is transformed with purified PCR fragments 116 to 119. PCR fragments 117 and 118 contain overlaps at their 5′ and 3′ ends and PCR fragments 116 and 119 contain overlaps at their 3′ and 5′ end respectively, such that this allows homologous recombination of all four PCR fragments. The 5′ end of PCR fragment 116 and the 3′ end of PCR fragment 119 are homologous to the INT1 locus and enables integration of all four PCR fragments in the INT1 locus (see FIG. 6). This results in one linear fragment consisting of PCR fragments 116 to 119 integrated in the INT1 locus. This method of integration is described in patent application WO2013076280. The INT1 locus is located at chromosome XV, 659 bp downstream of YOR071c and 998 bp upstream of YOR070c. This approach results in expression of the fumarate reductase protein of 1139 amino acids as indicated in SEQ ID NO: 8, which lacks the C-terminal amino acid SKI as compared to the native sequence from T. brucei.

Transformation mixtures are plated on YEPh-agar (per liter: 10 grams yeast extract, 20 grams PhytonePeptone, 20 grams galactose, 20 grams agar)) containing 100 μg nourseothricin (Jena Bioscience, Germany) per ml. After three days of growth at 30° C., individual transformants are re-streaked on YEPh-agar plates containing 20 grams galactose per liter and 100 μg nourseothricin per ml. Presence of the introduced genes is confirmed by using PCR using primer sequences that can anneal to the coding sequences of the ORF's encoded by SEQ ID NO: 38 and SEQ ID NO: 62.

Example 7: Transformation of Genes Encoding Malate Dehydrogenase Mutants to Strain REV-0001 and Production of Succinic Acid in Resulting Transformants

In order to determine if succinate levels are increased in strains expressing MDH mutants, MDH mutants are introduced in strain REV-0001 in which FRDg is expressed (Example 6). Based on the malic acid production results (Example 4) and the in vitro activity assay results (Example 5), 3 MDH mutants are selected: MUT_014, MUT_015 and MUT_034.

Generation of PCR Fragments

The amplification of PCR fragment 1, 2, 3, 4 and 5 is described in Example 2. In order to introduce the wild-type and diverse MDH mutants, PCR fragment 120 (wild-type MDH3, SEQ ID NO: 31), fragment 121 (MUT_014, SEQ ID NO: 64), fragment 122 (MUT_015, SEQ ID NO: 65) or fragment 123 (MUT_034, SEQ ID NO: 66) are used. The wild-type and mutant MDH3 genes are driven by the S. cerevisiae TDH3 terminator and termination is controlled by the S. cerevisiae GPM1 terminator. The cassettes are amplified by PCR using the primer sequences described in SEQ ID NO: 19 and SEQ ID NO: 20, to generate PCR fragments 120 to 123.

Primer sequences described in SEQ ID NO: 63 and SEQ ID NO: 41 are used to generate PCR fragment 124 consisting of the 3′ INT59 integration site, using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragments 1 to 5/120-123 and PCR fragment 124 are purified using the DNA Clean & Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA) according to manufacturer's instructions.

Transformation to REV-0001

Yeast transformation is performed by a method known by persons skilled in the art. S. cerevisiae strain REV-0001 is transformed with purified PCR fragments 1 to 5 and 124 in combination with PCR fragments 120, 121, 122 or 123. PCR fragments 2 to 5/120-123 contain overlaps at their 5′ and 3′ ends and PCR fragments 1 and 124 at their 3′ and 5′ end respectively, such that this allows homologous recombination of all seven PCR fragments. The 5′ end of PCR fragment 1 and the 3′ end of PCR fragment 124 are homologous to the INT59 locus and enable integration of all seven PCR fragments in the INT59 locus (see FIG. 7). This results in one linear fragment consisting of PCR fragments 2 to 5/120-123 integrated in the INT59 locus. This method of integration is described in patent application WO2013076280. The INT59 locus is located at chromosome XI, 923 bp downstream of YKR092C and 922 bp upstream of YKR093W. Transformation mixtures are plated on YEPh-agar (per liter: 10 grams yeast extract, 20 grams PhytonePeptone, 20 grams agar) containing 20 grams galactose per liter and 200 μg G418 (Sigma Aldrich, Zwijndrecht, The Netherlands) per ml. After three days of growth at 30° C., individual transformants are re-streaked on YEPh-agar plates containing 20 grams galactose per liter and 200 μg G418 per ml. Presence of all introduced genes is confirmed by PCR.

Dicarboxylic Acid Production

To determine dicarboxylic acid production, REV-0001-derived transformants expressing the succinic acid production pathway with either wild-type MDH3 or individual MDH mutants are grown in micro titer plates and dicarboxylic acid concentrations are determined as described in General Materials and Methods.

Succinic acid titers of strains expressing MUT_014, MUT_015 and MUT_34 are respectively 1.3-, 1.2 and 1.4-fold higher than strains expressing MDH3, indicating that succinic acid production is also improved in strains expressing MDH mutants.

Claims

1. A recombinant yeast host cell which is capable of producing a dicarboxylic acid and which comprises a nucleic acid sequence encoding a mutant polypeptide having malate dehydrogenase activity, wherein the mutant polypeptide comprises an amino acid sequence which, when aligned with the malate dehydrogenase comprising the sequence set out in SEQ ID NO: 39, comprises a mutation of an amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39, wherein the polypeptide has at least 60% sequence identity with SEQ ID NO: 39; and, wherein the mutant polypeptide has an increase in the nadp(H)- relative to nad(H)-dependent activity as compared to that of a reference polypeptide having nad(H)-dependent malate dehydrogenase activity (EC 1.1.1.37), and wherein the reference polypeptide has an aspartic acid (D) at amino acid 34.

2. The recombinant host cell according to claim 1, wherein the mutation of the amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39 is a substitution to a small amino acid.

3. The recombinant host cell according to claim 1, wherein the mutation of the amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39 is selected from the group of substitutions corresponding to 34G and 34S.

4. The recombinant host cell according to claim 1, wherein the mutant polypeptide having malate dehydrogenase activity further comprises a mutation of an amino acid residue corresponding to amino acid 36 in SEQ ID NO: 39.

5. The recombinant host cell according to claim 4, wherein the mutation of the amino acid residue corresponding to amino acid 36 in SEQ ID NO: 39 is selected from the group of substitutions corresponding to 36R, 36Q, 36A, 36E, 36P and 36S.

6. The recombinant host cell according to claim 1, wherein the amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39 is selected from glycine (G) or serine (S).

7. The recombinant host cell according to claim 1, wherein the nad(H)- and nadp(H)-dependent activities of the mutant polypeptide having malate dehydrogenase activity are both increased.

8. The recombinant host cell according to claim 1, wherein the mutant polypeptide having malate dehydrogenase activity is a mutant nad(H)-dependent malate dehydrogenase (EC 1.1.1.37).

9. The recombinant host cell according to claim 1, wherein the mutant polypeptide having malate dehydrogenase activity is a mutant peroxisomal nad(H)-dependent malate dehydrogenase.

10. The recombinant host cell according to claim 1, wherein the mutant polypeptide having malate dehydrogenase activity is a mutant of a homologous polypeptide having malate dehydrogenase activity.

11. The recombinant host cell according to claim 1, wherein the mutant polypeptide having malate dehydrogenase activity is a mutant nad(H)-dependent malate dehydrogenase from a yeast.

12. The recombinant yeast host cell according to claim 1, which is selected from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, Issatchenkia, Saccharomyces, Schizosaccharomyces, or Yarrowia strains.

13. The recombinant host cell according to claim 12, wherein the yeast cell is Saccharomyces cerevisiae.

14. The recombinant host cell according to claim 1, wherein the nucleic sequence encoding the mutant polypeptide having malate dehydrogenase activity is expressed in the cytosol and the mutant polypeptide having malate dehydrogenase activity is active in the cytosol.

15. The host cell according to claim 1, wherein the recombinant host cell further comprises one or more copies of a nucleic acid encoding one or more of a phosphoenolpyruvate carboxykinase, a phosphoenolpyruvate carboxylase, a pyruvate carboxylase, a fumarase, a fumarate reductase and/or a succinate transporter.

16. A method for production of a dicarboxylic acid, wherein the method comprises fermenting the recombinant host cell according to claim 1 under conditions suitable for production of the dicarboxylic acid.

17. The method according to claim 16, further comprising recovering the dicarboxylic acid from the fermentation medium.

18. The method according to claim 16, wherein the dicarboxylic acid is succinic acid, malic acid and/or fumaric acid.